Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having cellulolytic enhancing activity, catalytic domains, cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains or cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or cellulose binding domains.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 16/123,482 filed on Sep. 6, 2018, which is a divisional applicationof U.S. application Ser. No. 14/437,472 filed on Apr. 21, 2015, nowabandoned, which is a 35 U.S.C. § 371 national application ofPCT/US2013/065485 filed on Oct. 17, 2013, which claims priority or thebenefit under 35 U.S.C. § 119 of U.S. Provisional Application No.61/61/717,989 filed on Oct. 24, 2012, the contents of which are fullyincorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to polypeptides having cellulolyticenhancing activity, catalytic domains, and cellulose binding domains,and polynucleotides encoding the polypeptides, catalytic domains, andcellulose binding domains. The invention also relates to nucleic acidconstructs, vectors, and host cells comprising the polynucleotides aswell as methods of producing and using the polypeptides, catalyticdomains, and cellulose binding domains.

Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently linked bybeta-1,4-bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose. Once the cellulose is converted toglucose, the glucose can easily be fermented by yeast into ethanol.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin.

WO 2005/074647, WO 2008/148131, and WO 2011/035027 disclose GH61polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Thielavia terrestris. WO 2005/074656 and WO2010/065830 disclose GH61 polypeptides having cellulolytic enhancingactivity and the polynucleotides thereof from Thermoascus aurantiacus.WO 2007/089290 and WO 2012/149344 disclose GH61 polypeptides havingcellulolytic enhancing activity and the polynucleotides thereof fromTrichoderma reesei. WO 2009/085935, WO 2009/085859, WO 2009/085864, andWO 2009/085868 disclose GH61 polypeptides having cellulolytic enhancingactivity and the polynucleotides thereof from Myceliophthorathermophila. WO 2010/138754 discloses a GH61 polypeptide havingcellulolytic enhancing activity and the polynucleotide thereof fromAspergillus fumigatus. WO 2011/005867 discloses a GH61 polypeptidehaving cellulolytic enhancing activity and the polynucleotide thereoffrom Penicillium pinophilum. WO 2011/039319 discloses a GH61 polypeptidehaving cellulolytic enhancing activity and the polynucleotide thereoffrom Thermoascus sp. WO 2011/041397 discloses a GH61 polypeptide havingcellulolytic enhancing activity and the polynucleotide thereof fromPenicillium sp. WO 2011/041504 discloses GH61 polypeptides havingcellulolytic enhancing activity and the polynucleotides thereof fromThermoascus crustaceus. WO 2012/030799 discloses GH61 polypeptideshaving cellulolytic enhancing activity and the polynucleotides thereoffrom Aspergillus aculeatus. WO 2012/113340 discloses GH61 polypeptideshaving cellulolytic enhancing activity and the polynucleotides thereoffrom Thermomyces lanuginosus. WO 2012/122477 discloses GH61 polypeptideshaving cellulolytic enhancing activity and the polynucleotides thereoffrom Aurantiporus alborubescens, Trichophaea saccata, and Penicilliumthomii. WO 2012/135659 discloses a GH61 polypeptide having cellulolyticenhancing activity and the polynucleotide thereof from Talaromycesstipitatus. WO 2012/146171 discloses GH61 polypeptides havingcellulolytic enhancing activity and the polynucleotides thereof fromHumicola insolens. WO 2012/101206 discloses GH61 polypeptides havingcellulolytic enhancing activity and the polynucleotides thereof fromMaibranchea cinnamomea, Talaromyces leycettanus, and Chaetomiumthermophilum. WO 2013/043910 discloses GH61 polypeptides havingcellulolytic enhancing activity and the polynucleotides thereof fromAcrophialophora fusispora and Corynascus sepedonium. WO 2008/151043 andWO 2012/122518 disclose methods of increasing the activity of a GH61polypeptide having cellulolytic enhancing activity by adding a divalentmetal cation to a composition comprising the polypeptide.

There is a need in the art for new enzymes to increase efficiency and toprovide cost-effective enzyme solutions for saccharification ofcellulosic material.

The present invention provides GH61 polypeptides having cellulolyticenhancing activity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingcellulolytic enhancing activity selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 6, at least 65% sequence identity to themature polypeptide of SEQ ID NO: 8, at least 75% sequence identity tothe mature polypeptide of SEQ ID NO: 2, or at least 80% sequenceidentity to the mature polypeptide of SEQ ID NO: 4;

(b) a polypeptide encoded by a polynucleotide that hybridizes under atleast high stringency conditions with the mature polypeptide codingsequence of SEQ ID NO: 1 or the cDNA sequence thereof, the maturepolypeptide coding sequence of SEQ ID NO: 3 or the cDNA sequencethereof, the mature polypeptide coding sequence of SEQ ID NO: 5 or thecDNA sequence thereof, or the mature polypeptide coding sequence of SEQID NO: 7; or the full-length complement thereof;

(c) a polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 5 or the cDNA sequence thereof, at least 65% sequence identity tothe mature polypeptide coding sequence of SEQ ID NO: 7 or the cDNAsequence thereof, at least 75% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequencethereof, or at least 80% sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 3;

(d) a variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6, or SEQ ID NO: 8 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions; and

(e) a fragment of the polypeptide of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

The present invention also relates to isolated polypeptides comprising acatalytic domain selected from the group consisting of:

(a) a catalytic domain having at least 80% sequence identity to aminoacids 20 to 245 of SEQ ID NO: 2;

(b) a catalytic domain encoded by a polynucleotide that hybridizes underat least high stringency conditions with nucleotides 58 to 1122 of SEQID NO: 1; the cDNA sequence thereof; or the full-length complementthereof;

(c) a catalytic domain encoded by a polynucleotide having at least 80%sequence identity to nucleotides 58 to 1122 of SEQ ID NO: 1 or the cDNAsequence thereof;

(d) a variant of amino acids 20 to 245 of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and

(e) a fragment of the catalytic domain of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

The present invention also relates to isolated polypeptides comprising acellulose binding domain selected from the group consisting of:

(a) a cellulose binding domain having at least 80% sequence identity toamino acids 284 to 316 of SEQ ID NO: 2;

(b) a cellulose binding domain encoded by a polynucleotide thathybridizes under at least high stringency conditions with nucleotides1237 to 1458 of SEQ ID NO: 1; the cDNA sequence thereof; or thefull-length complement thereof;

(c) a cellulose binding domain encoded by a polynucleotide having atleast 80% sequence identity to nucleotides 1237 to 1458 of SEQ ID NO: 1or the cDNA sequence thereof;

(d) a variant of amino acids 284 to 316 of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and

(e) a fragment of the cellulose binding domain of (a), (b), (c), or (d)that has binding activity.

The present invention also relates to isolated polynucleotides encodingthe polypeptides of the present invention; nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides; and methods of producing the polypeptides.

The present invention also relates to processes for degrading acellulosic material, comprising: treating the cellulosic material withan enzyme composition in the presence of a polypeptide havingcellulolytic enhancing activity of the present invention.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more (e.g.,several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to processes of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more (e.g., several) fermenting microorganisms, wherein thecellulosic material is saccharified with an enzyme composition in thepresence of a polypeptide having cellulolytic enhancing activity of thepresent invention.

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to19 of SEQ ID NO: 2, amino acids 1 to 19 of SEQ ID NO: 4, amino acids 1to 19 of SEQ ID NO: 6, or amino acids 1 to 16 of SEQ ID NO: 8, which isoperably linked to a gene encoding a protein, wherein the protein isforeign to the signal peptide; nucleic acid constructs, expressionvectors, and recombinant host cells comprising the polynucleotides; andmethods of producing a protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of Lentinus similis GH61 polypeptides on thehydrolysis of microcrystalline cellulose at pH 5.0 by a composition ofTrichoderma reesei GH5 endoglucanase II and Aspergillus fumigatus GH3beta-glucosidase.

FIG. 2 shows the effect of Lentinus similis GH61 polypeptides on thehydrolysis of microcrystalline cellulose at pH 8.0 by a composition ofTrichoderma reesei GH5 endoglucanase II and Aspergillus fumigatus GH3beta-glucosidase.

FIG. 3 shows the activity of Lentinus similis P247JE GH61 polypeptide indegrading phosphoric acid-swollen cellulose (PASO) at pH 5.

FIG. 4 shows the activity of Lentinus similis P247JK GH61 polypeptide indegrading phosphoric acid-swollen cellulose (PASO) at pH 5.

FIG. 5 shows the activity of Lentinus similis P247JE GH61 polypeptide indegrading phosphoric acid-swollen cellulose (PASO) at pH 5.

FIG. 6 shows the effect of the Bulgaria inquinans GH61 polypeptide onthe hydrolysis of microcrystalline cellulose by a composition ofTrichoderma reesei GH5 endoglucanase II and Aspergillus fumigatus GH3beta-glucosidase.

FIG. 7 shows the effect of the Bulgaria inquinans GH61 polypeptide onthe hydrolysis of milled unwashed pretreated corn stover (PCS) by acellulolytic enzyme composition.

DEFINITIONS

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20 (polyoxyethylene sorbitan monolaurate). Oneunit of acetylxylan esterase is defined as the amount of enzyme capableof releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25°C.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.For purposes of the present invention, beta-glucosidase activity isdetermined using p-nitrophenyl-beta-D-glucopyranoside as substrateaccording to the procedure of Venturi et al., 2002, J. Basic Microbiol.42: 55-66. One unit of beta-glucosidase is defined as 1.0 μmole ofp-nitrophenolate anion produced per minute at 37° C., pH 5.0 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM succinicacid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl₂, 150 mM KCl,0.01% TRITON® X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethyleneglycol).

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. For purposes of the present invention,beta-xylosidase activity is determined using 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20 at pH 5, 40° C. One unit of beta-xylosidaseis defined as 1.0 μmole of p-nitrophenolate anion produced per minute at40° C., pH 5 from 1 mM p-nitrophenyl-beta-D-xyloside in 100 mM sodiumcitrate containing 0.01% TWEEN® 20.

Carbohydrate binding domain: The term “carbohydrate binding domain”means the region of an enzyme that mediates binding of the enzyme toamorphous regions of a cellulose substrate. The carbohydrate bindingdomain (CBD) is typically found either at the N-terminal or at theC-terminal extremity of an enzyme. The term “carbohydrate bindingdomain” is also referred herein as “cellulose binding domain”.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme. cDNA: The term“cDNA” means a DNA molecule that can be prepared by reversetranscription from a mature, spliced, mRNA molecule obtained from aeukaryotic or prokaryotic cell. cDNA lacks intron sequences that may bepresent in the corresponding genomic DNA. The initial, primary RNAtranscript is a precursor to mRNA that is processed through a series ofsteps, including splicing, before appearing as mature spliced mRNA.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing end(cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) of thechain (Teeri, 1997, Trends in Biotechnology 15: 160-167; Teeri et al.,1998, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity isdetermined according to the procedures described by Lever et al., 1972,Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters,149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187:283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. In thepresent invention, the Tomme et al. method can be used to determinecellobiohydrolase activity.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic enzyme activity include:(1) measuring the total cellulolytic enzyme activity, and (2) measuringthe individual cellulolytic enzyme activities (endoglucanases,cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al.,2006, Biotechnology Advances 24: 452-481. Total cellulolytic enzymeactivity can be measured using insoluble substrates, including WhatmanNo 1 filter paper, microcrystalline cellulose, bacterial cellulose,algal cellulose, cotton, pretreated lignocellulose, etc. The most commontotal cellulolytic activity assay is the filter paper assay usingWhatman No 1 filter paper as the substrate. The assay was established bythe International Union of Pure and Applied Chemistry (IUPAC) (Ghose,1987, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in production/release of sugarsduring hydrolysis of a cellulosic material by cellulolytic enzyme(s)under the following conditions: 1-50 mg of cellulolytic enzyme protein/gof cellulose in pretreated corn stover (PCS) (or other pretreatedcellulosic material) for 3-7 days at a suitable temperature such as 40°C.−80° C., e.g., 50° C., 55° C., 60° C., 65° C., or 70° C., and asuitable pH such as 4-9, e.g., 5.0, 5.5, 6.0, 6.5, or 7.0, compared to acontrol hydrolysis without addition of cellulolytic enzyme protein.Typical conditions are 1 ml reactions, washed or unwashed PCS, 5%insoluble solids (dry weight), 50 mM sodium acetate pH 5, 1 mM MnSO₄,50° C., 55° C., or 60° C., 72 hours, sugar analysis by AMINEX® HPX-87Hcolumn (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In one aspect, the cellulosicmaterial is any biomass material. In another aspect, the cellulosicmaterial is lignocellulose, which comprises cellulose, hemicelluloses,and lignin.

In an embodiment, the cellulosic material is agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, or wood (including forestryresidue).

In another embodiment, the cellulosic material is arundo, bagasse,bamboo, corn cob, corn fiber, corn stover, miscanthus, rice straw,switchgrass, or wheat straw.

In another embodiment, the cellulosic material is aspen, eucalyptus,fir, pine, poplar, spruce, or willow.

In another embodiment, the cellulosic material is algal cellulose,bacterial cellulose, cotton linter, filter paper, microcrystallinecellulose (e.g., AVICEL®), or phosphoric-acid treated cellulose.

In another embodiment, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Endoglucanase: The term “endoglucanase” means a4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3-1,4 glucans suchas cereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Biochem. J.316: 695-696. The enzymes in this family were originally classified as aglycoside hydrolase family based on measurement of very weakendo-1,4-beta-D-glucanase activity in one family member. GH61polypeptides are now classified as a lytic polysaccharide monooxygenase(Quinlan et al., 2011, Proc. Natl. Acad. Sci. USA 208: 15079-15084;Phillips et al., 2011, ACS Chem. Biol. 6: 1399-1406; Lin et al., 2012,Structure 20: 1051-1061) and placed into a new family designated“Auxiliary Activity 9” or “AA9”.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in naturalbiomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).Feruloyl esterase (FAE) is also known as ferulic acid esterase,hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA,cinnAE, FAE-I, or FAE-II. For purposes of the present invention,feruloyl esterase activity is determined using 0.5 mMp-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0. Oneunit of feruloyl esterase equals the amount of enzyme capable ofreleasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25° C.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has cellulolyticenhancing activity. In one aspect, a fragment contains at least 250amino acid residues, e.g., at least 265 amino acid residues or at least280 amino acid residues of SEQ ID NO: 2. In another aspect, a fragmentcontains at least 180 amino acid residues, e.g., at least 190 amino acidresidues or at least 200 amino acid residues of SEQ ID NO: 4. In anotheraspect, a fragment contains at least 205 amino acid residues, e.g., atleast 215 amino acid residues or at least 225 amino acid residues of SEQID NO: 6. In another aspect, a fragment contains at least 260 amino acidresidues, e.g., at least 275 amino acid residues or at least 290 aminoacid residues of SEQ ID NO: 8.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom andShoham, Current Opinion In Microbiology, 2003, 6(3): 219-228).Hemicellulases are key components in the degradation of plant biomass.Examples of hemicellulases include, but are not limited to, anacetylmannan esterase, an acetylxylan esterase, an arabinanase, anarabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase. The substrates for theseenzymes, hemicelluloses, are a heterogeneous group of branched andlinear polysaccharides that are bound via hydrogen bonds to thecellulose microfibrils in the plant cell wall, crosslinking them into arobust network. Hemicelluloses are also covalently attached to lignin,forming together with cellulose a highly complex structure. The variablestructure and organization of hemicelluloses require the concertedaction of many enzymes for its complete degradation. The catalyticmodules of hemicellulases are either glycoside hydrolases (GHs) thathydrolyze glycosidic bonds, or carbohydrate esterases (CEs), whichhydrolyze ester linkages of acetate or ferulic acid side groups. Thesecatalytic modules, based on homology of their primary sequence, can beassigned into GH and CE families. Some families, with an overall similarfold, can be further grouped into clans, marked alphabetically (e.g.,GH-A). A most informative and updated classification of these and othercarbohydrate active enzymes is available in the Carbohydrate-ActiveEnzymes (CAZy) database. Hemicellulolytic enzyme activities can bemeasured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59:1739-1752, at a suitable temperature such as 40° C.-80° C., e.g., 50°C., 55° C., 60° C., 65° C., or 70° C., and a suitable pH such as 4-9,e.g., 5.0, 5.5, 6.0, 6.5, or 7.0.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 0.2×SSC, 0.2% SDSat 65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance).

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 0.2×SSC, 0.2% SDSat 50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 20 to 316 of SEQ ID NO: 2 (P247JE) based onthe SignalP 3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340:783-795) that predicts amino acids 1 to 19 of SEQ ID NO: 2 are a signalpeptide. In another aspect, the mature polypeptide is amino acids 20 to231 of SEQ ID NO: 4 (P247JE) based on the SignalP 3.0 program thatpredicts amino acids 1 to 19 of SEQ ID NO: 4 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 20 to 254 of SEQID NO: 6 (P247JK) based on the SignalP 3.0 program that predicts aminoacids 1 to 19 of SEQ ID NO: 6 are a signal peptide. In another aspect,the mature polypeptide is amino acids 17 to 322 of SEQ ID NO: 8 (P24NHZ)based on the SignalP 3.0 program that predicts amino acids 1 to 16 ofSEQ ID NO: 8 are a signal peptide. It is known in the art that a hostcell may produce a mixture of two of more different mature polypeptides(i.e., with a different C-terminal and/or N-terminal amino acid)expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving cellulolytic enhancing activity. In one aspect, the maturepolypeptide coding sequence is nucleotides 58 to 1458 of SEQ ID NO: 1(D82AZVV) or the cDNA sequence thereof based on the SignalP 3.0 program(Bendtsen et al., 2004, supra) that predicts nucleotides 1 to 57 of SEQID NO: 1 encode a signal peptide. In another aspect, the maturepolypeptide coding sequence is nucleotides 58 to 985 of SEQ ID NO: 3(D82ATR) or the cDNA sequence thereof based on the SignalP 3.0 programthat predicts nucleotides 1 to 57 of SEQ ID NO: 3 encode a signalpeptide. In another aspect, the mature polypeptide coding sequence isnucleotides 58 to 1069 of SEQ ID NO: 5 (D82B12) or the cDNA sequencethereof based on the SignalP 3.0 program that predicts nucleotides 1 to57 of SEQ ID NO: 5 encode a signal peptide. In another aspect, themature polypeptide coding sequence is nucleotides 49 to 966 of SEQ IDNO: 7 (D134SM) based on the SignalP 3.0 program that predictsnucleotides 1 to 48 of SEQ ID NO: 7 encode a signal peptide.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5× SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that catalyzes the enhancement of the hydrolysis of acellulosic material by enzyme having cellulolytic activity. For purposesof the present invention, cellulolytic enhancing activity is determinedby measuring the increase in reducing sugars or the increase of thetotal of cellobiose and glucose from the hydrolysis of a cellulosicmaterial by cellulolytic enzyme under the following conditions: 1-50 mgof total protein/g of cellulose in pretreated corn stover (PCS), whereintotal protein is comprised of 50-99.5% w/w cellulolytic enzyme proteinand 0.5-50% w/w protein of a polypeptide having cellulolytic enhancingactivity for 1-7 days at a suitable temperature, such as 40° C.-80° C.,e.g., 50° C., 55° C., 60° C., 65° C., or 70° C., and a suitable pH, suchas 4-9, e.g., 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5, comparedto a control hydrolysis with equal total protein loading withoutcellulolytic enhancing activity (1-50 mg of cellulolytic protein/g ofcellulose in PCS).

GH61 polypeptide enhancing activity can be determined using a mixture ofCELLUCLAST® 1.5L (Novozymes A/S, Bagsværd, Denmark) in the presence of2-3% of total protein weight Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae according to WO 02/095014)or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase(recombinantly produced in Aspergillus oryzae as described in WO02/095014) of cellulase protein loading is used as the source of thecellulolytic activity.

GH61 polypeptide enhancing activity can also be determined by incubatingthe GH61 polypeptide with 0.5% phosphoric acid swollen cellulose (PASC),100 mM sodium acetate pH 5, 1 mM MnSO₄, 0.1% gallic acid, 0.025 mg/ml ofAspergillus fumigatus beta-glucosidase, and 0.01% TRITON® X-100 for24-96 hours at 40° C. followed by determination of the glucose releasedfrom the PASO

GH61 polypeptide enhancing activity can also be determined according toWO 2013/028928 for high temperature compositions.

GH61 polypeptide enhancing activity can also be determined according toExamples 11, 15, and 21 as described herein.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

The GH61 polypeptides of the present invention have at least 20%, e.g.,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, and at least 100% of the cellulolytic enhancingactivity of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, or SEQ ID NO: 8.

Pretreated corn stover: The term “Pretreated Corn Stover” or “PCS” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid, alkaline pretreatment, neutral pretreatment,or any pretreatment known in the art.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 3.0.0, 5.0.0 or later. The parametersused are gap open penalty of 10, gap extension penalty of 0.5, and theEBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the—nobrief option)is used as the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 3.0.0, 5.0.0 or later. The parameters used are gap open penaltyof 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version ofNCBI NUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the—nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having cellulolytic enhancing activity. In one aspect, asubsequence contains at least 750 nucleotides, e.g., at least 795nucleotides or at least 840 nucleotides of SEQ ID NO: 1. In anotheraspect, a subsequence contains at least 540 nucleotides, e.g., at least570 nucleotides or at least 600 nucleotides of SEQ ID NO: 3. In anotheraspect, a subsequence contains at least 615 nucleotides, e.g., at least645 nucleotides or at least 675 nucleotides of SEQ ID NO: 5. In oneaspect, a subsequence contains at least 780 nucleotides, e.g., at least825 nucleotides or at least 870 nucleotides of SEQ ID NO: 7.

Variant: The term “variant” means a polypeptide having cellulolyticenhancing activity comprising an alteration, i.e., a substitution,insertion, and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 45° C.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186: 1-67.

In the processes of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, 2006, Journal of the Science of Food and Agriculture 86(11):1636-1647; Spanikova and Biely, 2006, FEBS Letters 580(19): 4597-4601;Herrmann et al., 1997, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. A common total xylanolytic activity assay is based onproduction of reducing sugars from polymeric 4-O-methyl glucuronoxylanas described in Bailey, Biely, Poutanen, 1992, Interlaboratory testingof methods for assay of xylanase activity, Journal of Biotechnology23(3): 257-270. Xylanase activity can also be determined with 0.2%AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodiumphosphate pH 6 at 37° C. One unit of xylanase activity is defined as 1.0μmole of azurine produced per minute at 37° C., pH 6 from 0.2%AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37° C. One unit ofxylanase activity is defined as 1.0 μmole of azurine produced per minuteat 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mMsodium phosphate pH 6.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Cellulolytic Enhancing Activity

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 75%, e.g., at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%; the mature polypeptide of SEQ ID NO: 4 of atleast 80%, e.g., at least 81%, at least 82%, at least 83%, at least 84%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%; the mature polypeptide of SEQ ID NO: 6 of at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%; or the maturepolypeptide of SEQ ID NO: 8 of at least 65%, e.g., at least 70%, atleast 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%. In one aspect, the polypeptides differ by up to 10amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO:8.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,or SEQ ID NO: 8 or an allelic variant thereof; or is a fragment thereofhaving cellulolytic enhancing activity. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In another aspect,the polypeptide comprises or consists of amino acids 20 to 316 of SEQ IDNO: 2. In another aspect, the polypeptide comprises or consists of aminoacids 20 to 231 of SEQ ID NO: 4. In another aspect, the polypeptidecomprises or consists of amino acids 20 to 254 of SEQ ID NO: 6. Inanother aspect, the polypeptide comprises or consists of amino acids 17to 322 of SEQ ID NO: 8.

In another embodiment, the present invention relates to isolatedpolypeptides having cellulolytic enhancing activity encoded bypolynucleotides that hybridize under very low stringency conditions, lowstringency conditions, medium stringency conditions, medium-highstringency conditions, high stringency conditions, or very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, (ii) the cDNAsequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or (iii) thefull-length complement of (i) or (ii) (Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQID NO: 7, or a subsequence thereof, as well as the polypeptide of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, or a fragmentthereof, may be used to design nucleic acid probes to identify and cloneDNA encoding polypeptides having cellulolytic enhancing activity fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic DNA or cDNA of a cell of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 15, e.g., at least 25, atleast 35, or at least 70 nucleotides in length. Preferably, the nucleicacid probe is at least 100 nucleotides in length, e.g., at least 200nucleotides, at least 300 nucleotides, at least 400 nucleotides, atleast 500 nucleotides, at least 600 nucleotides, at least 700nucleotides, at least 800 nucleotides, or at least 900 nucleotides inlength. Both DNA and RNA probes can be used. The probes are typicallylabeled for detecting the corresponding gene (for example, with ³²P, ³H,³⁵S, biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having cellulolytic enhancing activity. Genomic orother DNA from such other strains may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that hybridizes with SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, the mature polypeptide codingsequence thereof, or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotides hybridize to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7; (ii)the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, or SEQ ID NO: 7; (iii) the cDNA sequence of SEQ ID NO: 1,SEQ ID NO: 3, or SEQ ID NO: 5; (iv) the full-length complement thereof;or (v) a subsequence thereof; under very low to very high stringencyconditions. Molecules to which the nucleic acid probe hybridizes underthese conditions can be detected using, for example, X-ray film or anyother detection means known in the art.

In one aspect, the nucleic acid probe is a polynucleotide that encodesthe polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ IDNO: 8; the mature polypeptide thereof; or a fragment thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, or SEQ ID NO: 7; the mature polypeptide coding sequence thereof; orthe cDNA sequence thereof.

In another embodiment, the present invention relates to isolatedpolypeptides having cellulolytic enhancing activity encoded bypolynucleotides having a sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 1 or the cDNA sequence thereof of at least75%, e.g., at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%; the mature polypeptide coding sequence of SEQ ID NO:3 or the cDNA sequence thereof of at least 80%, e.g., at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%; the mature polypeptidecoding sequence of SEQ ID NO: 5 or the cDNA sequence thereof of at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%; or themature polypeptide coding sequence of SEQ ID NO: 7 of at least at least65%, e.g., at least 70%, at least 75%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQID NO: 8 comprising a substitution, deletion, and/or insertion at one ormore (e.g., several) positions. In one aspect, the number of amino acidsubstitutions, deletions and/or insertions introduced into the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acidchanges may be of a minor nature, that is conservative amino acidsubstitutions or insertions that do not significantly affect the foldingand/or activity of the protein; small deletions, typically of 1-30 aminoacids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to 20-25residues; or a small extension that facilitates purification by changingnet charge or another function, such as a poly-histidine tract, anantigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R.L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide and/or thermal activity of the polypeptide, alter thesubstrate specificity, change the pH optimum, and the like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for cellulolytic enhancing activity to identifyamino acid residues that are critical to the activity of the molecule.See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The activesite of the enzyme or other biological interaction can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity ofessential amino acids can also be inferred from an alignment with arelated polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Cellulolytic Enhancing Activity

A polypeptide having cellulolytic enhancing activity of the presentinvention may be obtained from microorganisms of any genus. For purposesof the present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the polypeptide encodedby a polynucleotide is produced by the source or by a strain in whichthe polynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be a fungal polypeptide. In one aspect, thepolypeptide is a Lentinus polypeptide. In another aspect, thepolypeptide is a Lentinus similis polypeptide. In another aspect, thepolypeptide is a Bulgaria polypeptide. In another aspect, thepolypeptide is a Bulgaria inquinans polypeptide.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

A polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Catalytic Domains

In one embodiment, the present invention also relates to catalyticdomains having a sequence identity to amino acids 20 to 245 of SEQ IDNO: 2 of at least 80%, e.g., at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%. In one aspect, the catalytic domains comprise aminoacid sequences that differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, or 10, from amino acids 20 to 245 of SEQ ID NO: 2.

The catalytic domain preferably comprises or consists of amino acids 20to 245 of SEQ ID NO: 2; or an allelic variant thereof; or is a fragmentthereof having cellulolytic enhancing activity.

In another embodiment, the present invention also relates to catalyticdomains encoded by polynucleotides that hybridize under very lowstringency conditions, low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions (as defined above) withnucleotides 58 to 1122 of SEQ ID NO: 1; the cDNA sequence thereof; orthe full-length complement thereof (Sambrook et al., 1989, supra).

In another embodiment, the present invention also relates to catalyticdomains encoded by polynucleotides having a sequence identity tonucleotides 58 to 1122 of SEQ ID NO: 1, or the cDNA sequence thereof, ofat least 80%, e.g., at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%.

The polynucleotide encoding the catalytic domain preferably comprises orconsists of nucleotides 58 to 1122 of SEQ ID NO: 1; or the cDNA sequencethereof.

In another embodiment, the present invention also relates to catalyticdomain variants of amino acids 20 to 245 of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions. In one aspect, the number of amino acid substitutions,deletions and/or insertions introduced into the sequence of amino acids20 to 245 of SEQ ID NO: 2 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 8, 9, or10.

Binding Domains

In one embodiment, the present invention also relates to cellulosebinding domains having a sequence identity to amino acids 284 to 316 ofSEQ ID NO: 2 of at least 80%, e.g., at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%. In one aspect, the cellulose binding domainscomprise amino acid sequences that differ by up to 10 amino acids, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from amino acids 284 to 316 of SEQ IDNO: 2.

The cellulose binding domain preferably comprises or consists of aminoacids 284 to 316 of SEQ ID NO: 2; or an allelic variant thereof; or is afragment thereof having cellulose binding activity.

In another embodiment, the present invention also relates to cellulosebinding domains encoded by polynucleotides that hybridize under very lowstringency conditions, low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions (as defined above) withnucleotides 1237 to 1458 of SEQ ID NO: 1; the cDNA sequence thereof; orthe full-length complement thereof (Sambrook et al., 1989, supra).

In another embodiment, the present invention also relates to cellulosebinding domains encoded by polynucleotides having a sequence identity tonucleotides 1237 to 1458 of SEQ ID NO: 1, or the cDNA sequence thereof,of at least 80%, e.g., at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%.

The polynucleotide encoding the cellulose binding domain preferablycomprises or consists of nucleotides 1237 to 1458 of SEQ ID NO: 1 or thecDNA sequence thereof.

In another embodiment, the present invention also relates to cellulosebinding domain variants of amino acids 284 to 316 of SEQ ID NO: 2comprising a substitution, deletion, and/or insertion at one or more(e.g., several) positions. In one aspect, the number of amino acidsubstitutions, deletions and/or insertions introduced into the sequenceof amino acids 284 to 316 of SEQ ID NO: 2 is up to 10, e.g., 1, 2, 3, 4,5, 6, 8, 9, or 10.

A catalytic domain operably linked to the cellulose binding domain maybe from a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase,lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase, xylanase, or beta-xylosidase. The polynucleotideencoding the catalytic domain may be obtained from any prokaryotic,eukaryotic, or other source.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide, a catalytic domain, or cellulose binding domain of thepresent invention, as described herein.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well-known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligation activated transcription (LAT) andpolynucleotide-based amplification (NASBA) may be used. Thepolynucleotides may be cloned from a strain of Lentinus or Bulgaria, ora related organism and thus, for example, may be an allelic or speciesvariant of the polypeptide encoding region of the polynucleotide.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.These polypeptides may differ in some engineered way from thepolypeptide isolated from its native source, e.g., variants that differin specific activity, thermostability, pH optimum, or the like. Thevariants may be constructed on the basis of the polynucleotide presentedas the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, or SEQ ID NO: 7, or the cDNA sequence of SEQ ID NO: 1, SEQID NO: 3, or SEQ ID NO: 5, by introduction of nucleotide substitutionsthat do not result in a change in the amino acid sequence of thepolypeptide, but which correspond to the codon usage of the hostorganism intended for production of the enzyme, or by introduction ofnucleotide substitutions that may give rise to a different amino acidsequence. For a general description of nucleotide substitution, see,e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of the polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and mutant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus nigerglucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxam ide synthase), adeB(phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris an hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram positive or Gram negativebacterium. Gram positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp. 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and optionally (b) recovering thepolypeptide. In one aspect, the cell is a Lentinus cell. In anotheraspect, the cell is a Lentinus similis cell. In another aspect, the cellis a Bulgaria cell. In another aspect, the cell is a Bulgaria inquinanscell.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and optionally (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cells may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, a whole fermentation broth comprising thepolypeptide is recovered.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce a polypeptide ordomain in recoverable quantities. The polypeptide or domain may berecovered from the plant or plant part. Alternatively, the plant orplant part containing the polypeptide or domain may be used as such forimproving the quality of a food or feed, e.g., improving nutritionalvalue, palatability, and rheological properties, or to destroy anantinutritive factor

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide or domainmay be constructed in accordance with methods known in the art. Inshort, the plant or plant cell is constructed by incorporating one ormore expression constructs encoding the polypeptide or domain into theplant host genome or chloroplast genome and propagating the resultingmodified plant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide or domain operablylinked with appropriate regulatory sequences required for expression ofthe polynucleotide in the plant or plant part of choice.

Furthermore, the expression construct may comprise a selectable markeruseful for identifying plant cells into which the expression constructhas been integrated and DNA sequences necessary for introduction of theconstruct into the plant in question (the latter depends on the DNAintroduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide or domainis desired to be expressed (Sticklen, 2008, Nature Reviews 9: 433-443).For instance, the expression of the gene encoding a polypeptide ordomain may be constitutive or inducible, or may be developmental, stageor tissue specific, and the gene product may be targeted to a specifictissue or plant part such as seeds or leaves. Regulatory sequences are,for example, described by Tague et al., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide or domain in the plant. For instance, thepromoter enhancer element may be an intron that is placed between thepromoter and the polynucleotide encoding a polypeptide or domain. Forinstance, Xu et al., 1993, supra, disclose the use of the first intronof the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a polypeptide or domain canbe introduced into a particular plant variety by crossing, without theneed for ever directly transforming a plant of that given variety.Therefore, the present invention encompasses not only a plant directlyregenerated from cells which have been transformed in accordance withthe present invention, but also the progeny of such plants. As usedherein, progeny may refer to the offspring of any generation of a parentplant prepared in accordance with the present invention. Such progenymay include a DNA construct prepared in accordance with the presentinvention. Crossing results in the introduction of a transgene into aplant line by cross pollinating a starting line with a donor plant line.Non-limiting examples of such steps are described in U.S. Pat. No.7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideor domain of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide or domain under conditions conducive for production ofthe polypeptide or domain; and optionally (b) recovering the polypeptideor domain.

Removal or Reduction of Cellulolytic Enhancing Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotide, ora portion thereof, encoding a polypeptide of the present invention,which results in the mutant cell producing less of the polypeptide thanthe parent cell when cultivated under the same conditions.

The mutant cell may be constructed by reducing or eliminating expressionof the polynucleotide using methods well known in the art, for example,insertions, disruptions, replacements, or deletions. In a preferredaspect, the polynucleotide is inactivated. The polynucleotide to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required forexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thepolynucleotide. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the polynucleotide may be performed bysubjecting the parent cell to mutagenesis and selecting for mutant cellsin which expression of the polynucleotide has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the polynucleotide may also beaccomplished by insertion, substitution, or deletion of one or morenucleotides in the gene or a regulatory element required fortranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change in the open reading frame.Such modification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing thepolynucleotide to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of apolynucleotide is based on techniques of gene replacement, genedeletion, or gene disruption. For example, in the gene disruptionmethod, a nucleic acid sequence corresponding to the endogenouspolynucleotide is mutagenized in vitro to produce a defective nucleicacid sequence that is then transformed into the parent cell to produce adefective gene. By homologous recombination, the defective nucleic acidsequence replaces the endogenous polynucleotide. It may be desirablethat the defective polynucleotide also encodes a marker that may be usedfor selection of transformants in which the polynucleotide has beenmodified or destroyed. In an aspect, the polynucleotide is disruptedwith a selectable marker such as those described herein.

The present invention also relates to methods of inhibiting theexpression of a polypeptide having cellulolytic enhancing activity in acell, comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. In a preferredaspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA forinhibiting transcription. In another preferred aspect, the dsRNA ismicro RNA for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules, comprising a portion of the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7for inhibiting expression of the polypeptide in a cell. While thepresent invention is not limited by any particular mechanism of action,the dsRNA can enter a cell and cause the degradation of asingle-stranded RNA (ssRNA) of similar or identical sequences, includingendogenous mRNAs. When a cell is exposed to dsRNA, mRNA from thehomologous gene is selectively degraded by a process called RNAinterference (RNAi).

The dsRNAs of the present invention can be used in gene-silencing. Inone aspect, the invention provides methods to selectively degrade RNAusing a dsRNAi of the present invention. The process may be practiced invitro, ex vivo or in vivo. In one aspect, the dsRNA molecules can beused to generate a loss-of-function mutation in a cell, an organ or ananimal. Methods for making and using dsRNA molecules to selectivelydegrade RNA are well known in the art; see, for example, U.S. Pat. Nos.6,489,127; 6,506,559; 6,511,824; and 6,515,109.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a polynucleotide encoding thepolypeptide or a control sequence thereof or a silenced gene encodingthe polypeptide, which results in the mutant cell producing less of thepolypeptide or no polypeptide compared to the parent cell.

The polypeptide-deficient mutant cells are particularly useful as hostcells for expression of native and heterologous polypeptides. Therefore,the present invention further relates to methods of producing a nativeor heterologous polypeptide, comprising (a) cultivating the mutant cellunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide. The term “heterologous polypeptides” meanspolypeptides that are not native to the host cell, e.g., a variant of anative protein. The host cell may comprise more than one copy of apolynucleotide encoding the native or heterologous polypeptide.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallycellulolytic enhancing activity-free product are of particular interestin the production of eukaryotic polypeptides, in particular fungalproteins such as enzymes. The cellulolytic enhancing activity-deficientcells may also be used to express heterologous proteins ofpharmaceutical interest such as hormones, growth factors, receptors, andthe like. The term “eukaryotic polypeptides” includes not only nativepolypeptides, but also those polypeptides, e.g., enzymes, which havebeen modified by amino acid substitutions, deletions or additions, orother such modifications to enhance activity, thermostability, pHtolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from cellulolytic enhancing activity that is producedby a method of the present invention.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulationor a cell composition comprising a polypeptide of the present invention.The fermentation broth product further comprises additional ingredientsused in the fermentation process, such as, for example, cells(including, the host cells containing the gene encoding the polypeptideof the present invention which are used to produce the polypeptide),cell debris, biomass, fermentation media and/or fermentation products.In some embodiments, the composition is a cell-killed whole brothcontaining organic acid(s), killed cells and/or cell debris, and culturemedium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compositions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The fermentation broth formulations or cell compositions may furthercomprise multiple enzymatic activities, such as one or more (e.g.,several) enzymes selected from the group consisting of a cellulase, ahemicellulase, a CIP, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a catalase, a peroxidase, a protease,and a swollenin. The fermentation broth formulations or cellcompositions may also comprise one or more (e.g., several) enzymesselected from the group consisting of a hydrolase, an isomerase, aligase, a lyase, an oxidoreductase, or a transferase, e.g., analpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase,beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, glucoamylase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase, or xylanase.

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis (e.g., expression of cellulase and/or glucosidase enzyme(s)).In some embodiments, the cell-killed whole broth or composition containsthe spent cell culture medium, extracellular enzymes, and killedfilamentous fungal cells. In some embodiments, the microbial cellspresent in the cell-killed whole broth or composition can bepermeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Enzyme Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thecellulolytic enhancing activity of the composition has been increased,e.g., with an enrichment factor of at least 1.1.

The compositions may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the compositions may comprise multiple enzymaticactivities, such as one or more (e.g., several) enzymes selected fromthe group consisting of a cellulase, a hemicellulase, a CIP, anesterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, acatalase, a peroxidase, a protease, and a swollenin. The compositionsmay also comprise one or more (e.g., several) enzymes selected from thegroup consisting of a hydrolase, an isomerase, a ligase, a lyase, anoxidoreductase, or a transferase, e.g., an alpha-galactosidase,alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase,beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase,catalase, cellobiohydrolase, cellulase, chitinase, cutinase,cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase,esterase, glucoamylase, invertase, laccase, lipase, mannosidase,mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. The compositions may be stabilized in accordance withmethods known in the art.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Uses

The present invention is also directed to the following processes forusing the polypeptides having cellulolytic enhancing activity, orcompositions thereof.

The present invention also relates to processes for degrading acellulosic material, comprising: treating the cellulosic material withan enzyme composition in the presence of a polypeptide havingcellulolytic enhancing activity of the present invention. In one aspect,the processes further comprise recovering the degraded cellulosicmaterial. Soluble products of degradation of the cellulosic material canbe separated from insoluble cellulosic material using a method known inthe art such as, for example, centrifugation, filtration, or gravitysettling.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more (e.g.,several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to processes of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more (e.g., several) fermenting microorganisms, wherein thecellulosic material is saccharified with an enzyme composition in thepresence of a polypeptide having cellulolytic enhancing activity of thepresent invention. In one aspect, the fermenting of the cellulosicmaterial produces a fermentation product. In another aspect, theprocesses further comprise recovering the fermentation product from thefermentation.

The processes of the present invention can be used to saccharify thecellulosic material to fermentable sugars and to convert the fermentablesugars to many useful fermentation products, e.g., fuel (ethanol,n-butanol, isobutanol, biodiesel, jet fuel) and/or platform chemicals(e.g., acids, alcohols, ketones, gases, oils, and the like). Theproduction of a desired fermentation product from the cellulosicmaterial typically involves pretreatment, enzymatic hydrolysis(saccharification), and fermentation.

The processing of the cellulosic material according to the presentinvention can be accomplished using methods conventional in the art.Moreover, the processes of the present invention can be implementedusing any conventional biomass processing apparatus configured tooperate in accordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC), also sometimes calledconsolidated bioprocessing (CBP). SHF uses separate process steps tofirst enzymatically hydrolyze the cellulosic material to fermentablesugars, e.g., glucose, cellobiose, and pentose monomers, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the co-fermentation of multiple sugars (Sheehanand Himmel, 1999, Biotechnol. Prog. 15: 817-827). HHF involves aseparate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more (e.g.,several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd et al., 2002,Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood herein thatany method known in the art comprising pretreatment, enzymatichydrolysis (saccharification), fermentation, or a combination thereof,can be used in the practicing the processes of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (deCastilhos Corazza et al., 2003, Acta Scientiarum. Technology 25: 33-38;Gusakov and Sinitsyn, 1985, Enz. Microb. Technol. 7: 346-352), anattrition reactor (Ryu and Lee, 1983, Biotechnol. Bioeng. 25: 53-65).Additional reactor types include fluidized bed, upflow blanket,immobilized, and extruder type reactors for hydrolysis and/orfermentation.

Pretreatment. In practicing the processes of the present invention, anypretreatment process known in the art can be used to disrupt plant cellwall components of the cellulosic material (Chandra et al., 2007, Adv.Biochem. Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Adv.Biochem. Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009,Bioresource Technology 100: 10-18; Mosier et al., 2005, BioresourceTechnology 96: 673-686; Taherzadeh and Karimi, 2008, Int. J. Mol. Sci.9: 1621-1651; Yang and Wyman, 2008, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, sieving, pre-soaking, wetting, washing, and/or conditioningprior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, ionic liquid, and gammairradiation pretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, the cellulosic material isheated to disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. The cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably performed at 140-250° C., e.g., 160-200° C. or 170-190° C.,where the optimal temperature range depends on optional addition of achemical catalyst. Residence time for the steam pretreatment ispreferably 1-60 minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes,or 4-10 minutes, where the optimal residence time depends on thetemperature and optional addition of a chemical catalyst. Steampretreatment allows for relatively high solids loadings, so that thecellulosic material is generally only moist during the pretreatment. Thesteam pretreatment is often combined with an explosive discharge of thematerial after the pretreatment, which is known as steam explosion, thatis, rapid flashing to atmospheric pressure and turbulent flow of thematerial to increase the accessible surface area by fragmentation (Duffand Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi,2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent ApplicationNo. 2002/0164730). During steam pretreatment, hemicellulose acetylgroups are cleaved and the resulting acid autocatalyzes partialhydrolysis of the hemicellulose to monosaccharides and oligosaccharides.Lignin is removed to only a limited extent.

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Such a pretreatment can convertcrystalline cellulose to amorphous cellulose. Examples of suitablechemical pretreatment processes include, for example, dilute acidpretreatment, lime pretreatment, wet oxidation, ammonia fiber/freezeexpansion (AFEX), ammonia percolation (APR), ionic liquid, andorganosolv pretreatments.

A chemical catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 5% w/w) issometimes added prior to steam pretreatment, which decreases the timeand temperature, increases the recovery, and improves enzymatichydrolysis (Ballesteros et al., 2006, Appl. Biochem. Biotechnol.129-132: 496-508; Varga et al., 2004, Appl. Biochem. Biotechnol.113-116: 509-523; Sassner et al., 2006, Enzyme Microb. Technol. 39:756-762). In dilute acid pretreatment, the cellulosic material is mixedwith dilute acid, typically H₂SO₄, and water to form a slurry, heated bysteam to the desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource Technology 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to,sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), andammonia fiber/freeze expansion (AFEX) pretreatment.

Lime pretreatment is performed with calcium oxide or calcium hydroxideat temperatures of 85-150° C. and residence times from 1 hour to severaldays (Wyman et al., 2005, Bioresource Technology 96: 1959-1966; Mosieret al., 2005, Bioresource Technology 96: 673-686). WO 2006/110891, WO2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatmentmethods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technology 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed preferably at 1-40% drymatter, e.g., 2-30% dry matter or 5-20% dry matter, and often theinitial pH is increased by the addition of alkali such as sodiumcarbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion) can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber expansion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-150°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technology 96:2014-2018). During AFEX pretreatment cellulose and hemicelluloses remainrelatively intact. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose andlignin is removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. Biotechnol. 105-108: 69-85, and Mosier etal., 2005, Bioresource Technology 96: 673-686, and U.S. PublishedApplication 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as adilute acid treatment, and more preferably as a continuous dilute acidtreatment. The acid is typically sulfuric acid, but other acids can alsobe used, such as acetic acid, citric acid, nitric acid, phosphoric acid,tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.Mild acid treatment is conducted in the pH range of preferably 1-5,e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in therange from preferably 0.01 to 10 wt. % acid, e.g., 0.05 to 5 wt. % acidor 0.1 to 2 wt. % acid. The acid is contacted with the cellulosicmaterial and held at a temperature in the range of preferably 140-200°C., e.g., 165-190° C., for periods ranging from 1 to 60 minutes.

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt. %, e.g., 20-70 wt.% or 30-60 wt. %, such as around 40 wt. %. The pretreated cellulosicmaterial can be unwashed or washed using any method known in the art,e.g., washed with water.

Mechanical Pretreatment or Physical Pretreatment: The term “mechanicalpretreatment” or “physical pretreatment” refers to any pretreatment thatpromotes size reduction of particles. For example, such pretreatment caninvolve various types of grinding or milling (e.g., dry milling, wetmilling, or vibratory ball milling).

The cellulosic material can be pretreated both physically (mechanically)and chemically. Mechanical or physical pretreatment can be coupled withsteaming/steam explosion, hydrothermolysis, dilute or mild acidtreatment, high temperature, high pressure treatment, irradiation (e.g.,microwave irradiation), or combinations thereof. In one aspect, highpressure means pressure in the range of preferably about 100 to about400 psi, e.g., about 150 to about 250 psi. In another aspect, hightemperature means temperature in the range of about 100 to about 300°C., e.g., about 140 to about 200° C. In a preferred aspect, mechanicalor physical pretreatment is performed in a batch-process using a steamgun hydrolyzer system that uses high pressure and high temperature asdefined above, e.g., a Sunds Hydrolyzer available from Sunds DefibratorAB, Sweden. The physical and chemical pretreatments can be carried outsequentially or simultaneously, as desired.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto physical (mechanical) or chemical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms and/or enzymes (see, for example,Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh and Singh, 1993, Adv. Appl. Microbiol.39: 295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass:a review, in Enzymatic Conversion of Biomass for Fuels Production,Himmel, M. E., Baker, J. O., and Overend, R. P., eds., ACS SymposiumSeries 566, American Chemical Society, Washington, D.C., chapter 15;Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanolproduction from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996, Enz.Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990, Adv.Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification. In the hydrolysis step, also known assaccharification, the cellulosic material, e.g., pretreated, ishydrolyzed to break down cellulose and/or hemicellulose to fermentablesugars, such as glucose, cellobiose, xylose, xylulose, arabinose,mannose, galactose, and/or soluble oligosaccharides. In one aspect, thesugar is selected from the group consisting of glucose, xylose, mannose,galactose, and arabinose. The hydrolysis is performed enzymatically byan enzyme composition as described herein in the presence of apolypeptide having cellulolytic enhancing activity of the presentinvention. The enzymes of the compositions can be added simultaneouslyor sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzymes(s), i.e., optimalfor the enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the cellulosic material is fed gradually to,for example, an enzyme containing hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 120 hours, e.g., about 16 to about 72 hours or about24 to about 48 hours. The temperature is in the range of preferablyabout 25° C. to about 70° C., e.g., about 30° C. to about 65° C., about40° C. to about 60° C., or about 50° C. to about 55° C. The pH is in therange of preferably about 3 to about 8, e.g., about 3.5 to about 7,about 4 to about 6, or about 4.5 to about 5.5. The dry solids content isin the range of preferably about 5 to about 50 wt. %, e.g., about 10 toabout 40 wt. % or about 20 to about 30 wt. %.

The enzyme compositions can comprise any protein useful in degrading thecellulosic material.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins selected from the group consisting of acellulase, a hemicellulase, a CIP, an esterase, an expansin, a laccase,a ligninolytic enzyme, a pectinase, a catalase, a peroxidase, aprotease, and a swollenin. In another aspect, the cellulase ispreferably one or more (e.g., several) enzymes selected from the groupconsisting of an endoglucanase, a cellobiohydrolase, and abeta-glucosidase. In another aspect, the hemicellulase is preferably oneor more (e.g., several) enzymes selected from the group consisting of anacetylmannan esterase, an acetylxylan esterase, an arabinanase, anarabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase.

In another aspect, the enzyme composition comprises one or more (e.g.,several) cellulolytic enzymes. In another aspect, the enzyme compositioncomprises or further comprises one or more (e.g., several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes and one ormore (e.g., several) hemicellulolytic enzymes. In another aspect, theenzyme composition comprises one or more (e.g., several) enzymesselected from the group of cellulolytic enzymes and hemicellulolyticenzymes. In another aspect, the enzyme composition comprises anendoglucanase. In another aspect, the enzyme composition comprises acellobiohydrolase. In another aspect, the enzyme composition comprises acellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II. In another aspect, theenzyme composition comprises a beta-glucosidase. In another aspect, theenzyme composition comprises an endoglucanase and a cellobiohydrolase.In another aspect, the enzyme composition comprises an endoglucanase anda cellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II. In another aspect, theenzyme composition comprises an endoglucanase and a beta-glucosidase. Inanother aspect, the enzyme composition comprises a beta-glucosidase anda cellobiohydrolase. In another aspect, the enzyme composition comprisesa beta-glucosidase and a cellobiohydrolase I, a cellobiohydrolase II, ora combination of a cellobiohydrolase I and a cellobiohydrolase II. Inanother aspect, the enzyme composition comprises an endoglucanase, abeta-glucosidase, and a cellobiohydrolase. In another aspect, the enzymecomposition comprises an endoglucanase, a beta-glucosidase, and acellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetylxylan esterase. In another aspect, the enzyme compositioncomprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect,the enzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase). Inanother aspect, the enzyme composition comprises a xylanase. In anembodiment, the xylanase is a Family 10 xylanase. In another embodiment,the xylanase is a Family 11 xylanase. In another aspect, the enzymecomposition comprises a xylosidase (e.g., beta-xylosidase).

In another aspect, the enzyme composition comprises a CIP. In anotheraspect, the enzyme composition comprises an esterase. In another aspect,the enzyme composition comprises an expansin. In another aspect, theenzyme composition comprises a laccase. In another aspect, the enzymecomposition comprises a ligninolytic enzyme. In a preferred aspect, theligninolytic enzyme is a manganese peroxidase. In another preferredaspect, the ligninolytic enzyme is a lignin peroxidase. In anotherpreferred aspect, the ligninolytic enzyme is a H₂O₂-producing enzyme. Inanother aspect, the enzyme composition comprises a pectinase. In anotheraspect, the enzyme composition comprises a catalase. In another aspect,the enzyme composition comprises a peroxidase. In another aspect, theenzyme composition comprises a protease. In another aspect, the enzymecomposition comprises a swollenin.

In the processes of the present invention, the enzyme(s) can be addedprior to or during saccharification, saccharification and fermentation,or fermentation.

One or more (e.g., several) components of the enzyme composition may benative proteins, recombinant proteins, or a combination of nativeproteins and recombinant proteins. For example, one or more (e.g.,several) components may be native proteins of a cell, which is used as ahost cell to express recombinantly one or more (e.g., several) othercomponents of the enzyme composition. It is understood herein that therecombinant proteins may be heterologous (e.g., foreign) and/or nativeto the host cell. One or more (e.g., several) components of the enzymecomposition may be produced as monocomponents, which are then combinedto form the enzyme composition. The enzyme composition may be acombination of multicomponent and monocomponent protein preparations.

The enzymes used in the processes of the present invention may be in anyform suitable for use, such as, for example, a fermentation brothformulation or a cell composition, a cell lysate with or withoutcellular debris, a semi-purified or purified enzyme preparation, or ahost cell as a source of the enzymes. The enzyme composition may be adry powder or granulate, a non-dusting granulate, a liquid, a stabilizedliquid, or a stabilized protected enzyme. Liquid enzyme preparationsmay, for instance, be stabilized by adding stabilizers such as a sugar,a sugar alcohol or another polyol, and/or lactic acid or another organicacid according to established processes.

The optimum amounts of the enzymes and a polypeptide having cellulolyticenhancing activity depend on several factors including, but not limitedto, the mixture of cellulolytic enzymes and/or hemicellulolytic enzymes,the cellulosic material, the concentration of cellulosic material, thepretreatment(s) of the cellulosic material, temperature, time, pH, andinclusion of a fermenting organism (e.g., for SimultaneousSaccharification and Fermentation).

In one aspect, an effective amount of cellulolytic or hemicellulolyticenzyme to the cellulosic material is about 0.5 to about 50 mg, e.g.,about 0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5to about 10 mg per g of the cellulosic material.

In another aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to the cellulosic material is about 0.01to about 50.0 mg, e.g., about 0.01 to about 40 mg, about 0.01 to about30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01to about 5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg,about 0.075 to about 1.25 mg, about 0.1 to about 1.25 mg, about 0.15 toabout 1.25 mg, or about 0.25 to about 1.0 mg per g of the cellulosicmaterial.

In another aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to cellulolytic or hemicellulolyticenzyme is about 0.005 to about 1.0 g, e.g., about 0.01 to about 1.0 g,about 0.15 to about 0.75 g, about 0.15 to about 0.5 g, about 0.1 toabout 0.5 g, about 0.1 to about 0.25 g, or about 0.05 to about 0.2 g perg of cellulolytic or hemicellulolytic enzyme.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the cellulosic material (collectively hereinafter“polypeptides having enzyme activity”) can be derived or obtained fromany suitable origin, including, archaeal, bacterial, fungal, yeast,plant, or animal origin. The term “obtained” also means herein that theenzyme may have been produced recombinantly in a host organism employingmethods described herein, wherein the recombinantly produced enzyme iseither native or foreign to the host organism or has a modified aminoacid sequence, e.g., having one or more (e.g., several) amino acids thatare deleted, inserted and/or substituted, i.e., a recombinantly producedenzyme that is a mutant and/or a fragment of a native amino acidsequence or an enzyme produced by nucleic acid shuffling processes knownin the art. Encompassed within the meaning of a native enzyme arenatural variants and within the meaning of a foreign enzyme are variantsobtained by, e.g., site-directed mutagenesis or shuffling.

A polypeptide having enzyme activity may be a bacterial polypeptide. Forexample, the polypeptide may be a Gram positive bacterial polypeptidesuch as a Bacillus, Streptococcus, Streptomyces, Staphylococcus,Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus,Caldicellulosiruptor, Acidothermus, Thermobifidia, or Oceanobacilluspolypeptide having enzyme activity, or a Gram negative bacterialpolypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, orUreaplasma polypeptide having enzyme activity.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide having enzyme activity.

The polypeptide having enzyme activity may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having enzyme activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having enzymeactivity.

In one aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide having enzyme activity.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum,Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporiummerdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielaviaspededonium, Thielavia setosa, Thielavia subthermophila, Thielaviaterrestris, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaeasaccata polypeptide having enzyme activity.

Chemically modified or protein engineered mutants of polypeptides havingenzyme activity may also be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host can be a heterologous host (enzyme is foreign tohost), but the host may under certain conditions also be a homologoushost (enzyme is native to host). Monocomponent cellulolytic proteins mayalso be prepared by purifying such a protein from a fermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC® CTec (Novozymes A/S),CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S),CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), SPEZYME™ CP(Genencor Int.), ACCELERASE™ TRIO (DuPont), FILTRASE® NL (DSM);METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (Röhm GmbH), or ALTERNAFUEL®CMAX3™ (Dyadic International, Inc.). The cellulolytic enzyme preparationis added in an amount effective from about 0.001 to about 5.0 wt. % ofsolids, e.g., about 0.025 to about 4.0 wt. % of solids or about 0.005 toabout 2.0 wt. % of solids.

Examples of bacterial endoglucanases that can be used in the processesof the present invention, include, but are not limited to, Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655; WO 00/70031; WO05/093050), Erwinia carotovara endoglucanase (Saarilahti et al., 1990,Gene 90: 9-14), Thermobifida fusca endoglucanase III (WO 05/093050), andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention, include, but are not limited to, Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichodermareesei Cel7B endoglucanase I (GenBank:M15665), Trichoderma reeseiendoglucanase II (Saloheimo et al., 1988, Gene 63:11-22), Trichodermareesei Cel5A endoglucanase II (GenBank:M19373), Trichoderma reeseiendoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64:555-563, GenBank:AB003694), Trichoderma reesei endoglucanase V(Saloheimo et al., 1994, Molecular Microbiology 13: 219-228,GenBank:Z33381), Aspergillus aculeatus endoglucanase (Ooi et al., 1990,Nucleic Acids Research 18: 5884), Aspergillus kawachii endoglucanase(Sakamoto et al., 1995, Current Genetics 27: 435-439), Fusariumoxysporum endoglucanase (GenBank:L29381), Humicola grisea var.thermoidea endoglucanase (GenBank:AB003107), Melanocarpus albomycesendoglucanase (GenBank:MAL515703), Neurospora crassa endoglucanase(GenBank:XM_324477), Humicola insolens endoglucanase V, Myceliophthorathermophila CBS 117.65 endoglucanase, Thermoascus aurantiacusendoglucanase I (GenBank:AF487830) and Trichoderma reesei strain No.VTT-D-80133 endoglucanase (Gen Bank: M15665).

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO2011/059740), Chaetomium thermophilum cellobiohydrolase I, Chaetomiumthermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolaseI, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871),Penicillium occitanis cellobiohydrolase I (GenBank:AY690482),Talaromyces emersonii cellobiohydrolase I (GenBank:AF439936), Thielaviahyrcanie cellobiohydrolase II (WO 2010/141325), Thielavia terrestriscellobiohydrolase II (CEL6A, WO 2006/074435), Trichoderma reeseicellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, andTrichophaea saccata cellobiohydrolase II (WO 2010/057086).

Examples of beta-glucosidases useful in the present invention include,but are not limited to, beta-glucosidases from Aspergillus aculeatus(Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus fumigatus (WO2005/047499), Aspergillus niger (Dan et al., 2000, J. Biol. Chem. 275:4973-4980), Aspergillus oryzae (WO 02/095014), Penicillium brasilianumIBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO2011/035029), and Trichophaea saccata (WO 2007/019442).

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is an Aspergillus oryzae beta-glucosidase variant BGfusion protein (WO 2008/057637) or an Aspergillus oryzaebeta-glucosidase fusion protein (WO 2008/057637).

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat, 1991, Biochem. J. 280: 309-316,and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO99/10481, WO 99/025847, WO 99/031255, WO 2002/101078, WO 2003/027306, WO2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO2008/008070, WO 2008/008793, U.S. Pat. Nos. 5,457,046, 5,648,263, and5,686,593.

In one aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a soluble activating divalent metalcation according to WO 2008/151043, e.g., manganese or copper.

In another aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a dioxy compound, a bicyliccompound, a heterocyclic compound, a nitrogen-containing compound, aquinone compound, a sulfur-containing compound, or a liquor obtainedfrom a pretreated cellulosic material such as pretreated corn stover(PCS).

The dioxy compound may include any suitable compound containing two ormore oxygen atoms. In some aspects, the dioxy compounds contain asubstituted aryl moiety as described herein. The dioxy compounds maycomprise one or more (e.g., several) hydroxyl and/or hydroxylderivatives, but also include substituted aryl moieties lacking hydroxyland hydroxyl derivatives. Non-limiting examples of the dioxy compoundsinclude pyrocatechol or catechol; caffeic acid; 3,4-dihydroxybenzoicacid; 4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid;methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone;2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid;4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid; ethylgallate; methyl glycolate; dihydroxyfumaric acid; 2-butyne-1,4-diol;(croconic acid; 1,3-propanediol; tartaric acid; 2,4-pentanediol;3-ethyoxy-1,2-propanediol; 2,4,4′-trihydroxybenzophenone;cis-2-butene-1,4-diol; 3,4-dihydroxy-3-cyclobutene-1,2-dione;dihydroxyacetone; acrolein acetal; methyl-4-hydroxybenzoate;4-hydroxybenzoic acid; and methyl-3,5-dimethoxy-4-hydroxybenzoate; or asalt or solvate thereof.

The bicyclic compound may include any suitable substituted fused ringsystem as described herein. The compounds may comprise one or more(e.g., several) additional rings, and are not limited to a specificnumber of rings unless otherwise stated. In one aspect, the bicycliccompound is a flavonoid. In another aspect, the bicyclic compound is anoptionally substituted isoflavonoid. In another aspect, the bicycliccompound is an optionally substituted flavylium ion, such as anoptionally substituted anthocyanidin or optionally substitutedanthocyanin, or derivative thereof. Non-limiting examples of thebicyclic compounds include epicatechin; quercetin; myricetin; taxifolin;kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin;cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.

The heterocyclic compound may be any suitable compound, such as anoptionally substituted aromatic or non-aromatic ring comprising aheteroatom, as described herein. In one aspect, the heterocyclic is acompound comprising an optionally substituted heterocycloalkyl moiety oran optionally substituted heteroaryl moiety. In another aspect, theoptionally substituted heterocycloalkyl moiety or optionally substitutedheteroaryl moiety is an optionally substituted 5-memberedheterocycloalkyl or an optionally substituted 5-membered heteroarylmoiety. In another aspect, the optionally substituted heterocycloalkylor optionally substituted heteroaryl moiety is an optionally substitutedmoiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl,oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl,thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl,carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl,quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl,dimethylhydantoin, pyrazinyl, tetrahydrofuranyl, pyrrolinyl,pyrrolidinyl, morpholinyl, indolyl, diazepinyl, azepinyl, thiepinyl,piperidinyl, and oxepinyl. In another aspect, the optionally substitutedheterocycloalkyl moiety or optionally substituted heteroaryl moiety isan optionally substituted furanyl. Non-limiting examples of theheterocyclic compounds include(1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one;4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2(5H)-furanone;[1,2-dihydroxyethyl]furan-2,3,4(5H)-trione; α-hydroxy-γ-butyrolactone;ribonic γ-lactone; aldohexuronicaldohexuronic acid γ-lactone; gluconicacid δ-lactone; 4-hydroxycoumarin; dihydrobenzofuran;5-(hydroxymethyl)furfural; furoin; 2(5H)-furanone;5,6-dihydro-2H-pyran-2-one; and5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvatethereof.

The nitrogen-containing compound may be any suitable compound with oneor more nitrogen atoms. In one aspect, the nitrogen-containing compoundcomprises an amine, imine, hydroxylamine, or nitroxide moiety.Non-limiting examples of the nitrogen-containing compounds includeacetone oxime; violuric acid; pyridine-2-aldoxime; 2-aminophenol;1,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy;5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterine; andmaleamic acid; or a salt or solvate thereof.

The quinone compound may be any suitable compound comprising a quinonemoiety as described herein. Non-limiting examples of the quinonecompounds include 1,4-benzoquinone; 1,4-naphthoquinone;2-hydroxy-1,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1,4-benzoquinone orcoenzyme Q₀; 2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone;1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione oradrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone; pyrroloquinolinequinone; or a salt or solvate thereof.

The sulfur-containing compound may be any suitable compound comprisingone or more sulfur atoms. In one aspect, the sulfur-containing comprisesa moiety selected from thionyl, thioether, sulfinyl, sulfonyl,sulfamide, sulfonamide, sulfonic acid, and sulfonic ester. Non-limitingexamples of the sulfur-containing compounds include ethanethiol;2-propanethiol; 2-propene-1-thiol; 2-mercaptoethanesulfonic acid;benzenethiol; benzene-1,2-dithiol; cysteine; methionine; glutathione;cystine; or a salt or solvate thereof.

In one aspect, an effective amount of such a compound described above tocellulosic material as a molar ratio to glucosyl units of cellulose isabout 10⁻⁶ to about 10, e.g., about 10⁻⁶ to about 7.5, about 10⁻⁶ toabout 5, about 10⁻⁶ to about 2.5, about 10⁻⁶ to about 1, about 10⁻⁵ toabout 1, about 10⁻⁵ to about 10⁻¹, about 10⁻⁴ to about 10⁻¹, about 10⁻³to about 10⁻¹, or about 10⁻³ to about 10⁻². In another aspect, aneffective amount of such a compound described above is about 0.1 μM toabout 1 M, e.g., about 0.5 μM to about 0.75 M, about 0.75 μM to about0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M, about 5 μMto about 50 mM, about 10 μM to about 25 mM, about 50 μM to about 25 mM,about 10 μM to about 10 mM, about 5 μM to about 5 mM, or about 0.1 mM toabout 1 mM.

The term “liquor” means the solution phase, either aqueous, organic, ora combination thereof, arising from treatment of a lignocellulose and/orhemicellulose material in a slurry, or monosaccharides thereof, e.g.,xylose, arabinose, mannose, etc., under conditions as described herein,and the soluble contents thereof. A liquor for cellulolytic enhancementof a GH61 polypeptide can be produced by treating a lignocellulose orhemicellulose material (or feedstock) by applying heat and/or pressure,optionally in the presence of a catalyst, e.g., acid, optionally in thepresence of an organic solvent, and optionally in combination withphysical disruption of the material, and then separating the solutionfrom the residual solids. Such conditions determine the degree ofcellulolytic enhancement obtainable through the combination of liquorand a GH61 polypeptide during hydrolysis of a cellulosic substrate by acellulase preparation. The liquor can be separated from the treatedmaterial using a method standard in the art, such as filtration,sedimentation, or centrifugation.

In one aspect, an effective amount of the liquor to cellulose is about10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about 7.5 g,about 10⁻⁶ to about 5 g, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ to about1 g, about 10⁻⁵ to about 1 g, about 10⁻⁵ to about 10⁻¹ g, about 10⁻⁴ toabout 10⁻¹ g, about 10⁻³ to about 10⁻¹ g, or about 10⁻³ to about 10⁻² gper g of cellulose.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), CELLIC®HTec3 (Novozymes A/S), VISCOZYME® (Novozymes A/S), ULTRAFLO® (NovozymesA/S), PULPZYME® HC (Novozymes A/S), MULTIFECT® Xylanase (Genencor),ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A(AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit,Wales, UK), DEPOL™ 740L. (Biocatalysts Limit, Wales, UK), DEPOL™ 762P(Biocatalysts Limit, Wales, UK), ALTERNA FUEL 100P (Dyadic), and ALTERNAFUEL 200P (Dyadic).

Examples of xylanases useful in the processes of the present inventioninclude, but are not limited to, xylanases from Aspergillus aculeatus(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp.(WO 2010/126772), Talaromyces lanuginosus GH11 (WO 2012/130965),Talaromyces thermophilus GH11 (WO 2012/13095), Thielavia terrestris NRRL8126 (WO 2009/079210), and Trichophaea saccata GH10 (WO 2011/057083).

Examples of beta-xylosidases useful in the processes of the presentinvention include, but are not limited to, beta-xylosidases fromNeurospora crassa (SwissProt:Q7SOW4), Trichoderma reesei(UniProtKB/TrEMBL:Q92458), Talaromyces emersonii (SwissProt:Q8X212), andTalaromyces thermophilus GH11 (WO 2012/13095).

Examples of acetylxylan esterases useful in the processes of the presentinvention include, but are not limited to, acetylxylan esterases fromAspergillus aculeatus (WO 2010/108918), Chaetomium globosum(UniProt:Q2GWX4), Chaetomium gracile (GeneSeqP:AAB82124), Humicolainsolens DSM 1800 (WO 2009/073709), Hypocrea jecorina (WO 2005/001036),Myceliophtera thermophila (WO 2010/014880), Neurospora crassa(UniProt:q7s259), Phaeosphaeria nodorum (UniProt:QOUHJ1), and Thielaviaterrestris NRRL 8126 (WO 2009/042846).

Examples of feruloyl esterases (ferulic acid esterases) useful in theprocesses of the present invention include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122),Neosartorya fischeri (UniProt:A1D9T4), Neurospora crassa(UniProt:Q9HGR3), Penicillium aurantiogriseum (WO 2009/127729), andThielavia terrestris (WO 2010/053838 and WO 2010/065448).

Examples of arabinofuranosidases useful in the processes of the presentinvention include, but are not limited to, arabinofuranosidases fromAspergillus niger (GeneSeqP:AAR94170), Humicola insolens DSM 1800 (WO2006/114094 and WO 2009/073383), and M. giganteus (WO 2006/114094).

Examples of alpha-glucuronidases useful in the processes of the presentinvention include, but are not limited to, alpha-glucuronidases fromAspergillus clavatus (UniProt:alcc12), Aspergillus fumigatus(SwissProt:Q4WW45), Aspergillus niger (U ni Prot: Q96WX9), Aspergillusterreus (Swiss Prot: Q0CJP9), Humicola insolens (WO 2010/014706),Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii(UniProt:Q8X211), and Trichoderma reesei (UniProt:Q99024).

In a preferred embodiment, the enzyme composition is a high temperaturecomposition, i.e., a composition that is able to hydrolyze a cellulosicmaterial in the range of about 55° C. to about 70° C. In anotherpreferred embodiment, the enzyme composition is a high temperaturecomposition, i.e., a composition that is able to hydrolyze a cellulosicmaterial at a temperature of about 55° C., about 56° C., about 57° C.,about 58° C., about 59° C., about 60° C., about 61° C., about 62° C.,about 63° C., about 64° C., about 65° C., about 66° C., about 67° C.,about 68° C., about 69° C., or about 70° C. In another preferredembodiment, the enzyme composition is a high temperature composition,i.e., a composition that is able to hydrolyze a cellulosic material at atemperature of at least 55° C., at least 56° C., at least 57° C., atleast 58° C., at least 59° C., at least 60° C., at least 61° C., atleast 62° C., at least 63° C., at least 64° C., at least 65° C., atleast 66° C., at least 67° C., at least 68° C., at least 69° C., or atleast 70° C.

In another preferred embodiment, the enzyme composition is a hightemperature composition as disclosed in WO 2011/057140, which isincorporated herein in its entirety by reference.

The polypeptides having enzyme activity used in the processes of thepresent invention may be produced by fermentation of the above-notedmicrobial strains on a nutrient medium containing suitable carbon andnitrogen sources and inorganic salts, using procedures known in the art(see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, C A, 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand enzyme production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, N Y, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

Fermentation. The fermentable sugars obtained from the hydrolyzedcellulosic material can be fermented by one or more (e.g., several)fermenting microorganisms capable of fermenting the sugars directly orindirectly into a desired fermentation product. “Fermentation” or“fermentation process” refers to any fermentation process or any processcomprising a fermentation step. Fermentation processes also includefermentation processes used in the consumable alcohol industry (e.g.,beer and wine), dairy industry (e.g., fermented dairy products), leatherindustry, and tobacco industry. The fermentation conditions depend onthe desired fermentation product and fermenting organism and can easilybe determined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on economics, i.e., costs per equivalent sugarpotential, and recalcitrance to enzymatic conversion.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be hexose and/or pentose fermenting organisms, or acombination thereof. Both hexose and pentose fermenting organisms arewell known in the art. Suitable fermenting microorganisms are able toferment, i.e., convert, sugars, such as glucose, xylose, xylulose,arabinose, maltose, mannose, galactose, and/or oligosaccharides,directly or indirectly into the desired fermentation product. Examplesof bacterial and fungal fermenting organisms producing ethanol aredescribed by Lin et al., 2006, Appl. MicrobioL Biotechnol. 69: 627-642.

Examples of fermenting microorganisms that can ferment hexose sugarsinclude bacterial and fungal organisms, such as yeast. Yeast includestrains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candidasonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Xylose fermenting yeast include strains of Candida, preferably C.sheatae or C. sonorensis; and strains of Pichia, e.g., P. stipitis, suchas P. stipitis CBS 5773. Pentose fermenting yeast include strains ofPachysolen, preferably P. tannophilus. Organisms not capable offermenting pentose sugars, such as xylose and arabinose, may begenetically modified to do so by methods known in the art.

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, 1996, supra).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include,e.g., BIOFERM™ AFT and XR (NABC—North American Bioproducts Corporation,GA, USA), ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™(Fleischmann's Yeast, USA), FERMIOL™ (DSM Specialties), GERT STRAND™(Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC™ fresh yeast(Ethanol Technology, WI, USA).

In an aspect, the fermenting microorganism has been genetically modifiedto provide the ability to ferment pentose sugars, such as xyloseutilizing, arabinose utilizing, and xylose and arabinose co-utilizingmicroorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Appl.Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Appl. Environ.Microbiol. 64: 1852-1859; Kotter and Ciriacy, 1993, Appl. Microbiol.Biotechnol. 38: 776-783; Walfridsson et al., 1995, Appl. Environ.Microbiol. 61: 4184-4190; Kuyper et al., 2004, FEMS Yeast Research 4:655-664; Beall et al., 1991, Biotech. Bioeng. 38: 296-303; Ingram etal., 1998, Biotechnol. Bioeng. 58: 204-214; Zhang et al., 1995, Science267: 240-243; Deanda et al., 1996, Appl. Environ. Microbiol. 62:4465-4470; WO 03/062430).

In one aspect, the fermenting organism comprises an isolatedpolynucleotide encoding a polypeptide having cellulolytic enhancingactivity of the present invention.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedcellulosic material or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, e.g., about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., e.g.,about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6,or 7.

In one aspect, the yeast and/or another microorganism are applied to thedegraded cellulosic material and the fermentation is performed for about12 to about 96 hours, such as typically 24-60 hours. In another aspect,the temperature is preferably between about 20° C. to about 60° C.,e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., orabout 32° C. to about 50° C., and the pH is generally from about pH 3 toabout pH 7, e.g., about pH 4 to about pH 7. However, some fermentingorganisms, e.g., bacteria, have higher fermentation temperature optima.Yeast or another microorganism is preferably applied in amounts ofapproximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰,especially approximately 2×10⁸ viable cell count per ml of fermentationbroth. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation products: A fermentation product can be any substancederived from the fermentation. The fermentation product can be, withoutlimitation, an alcohol (e.g., arabinitol, n-butanol, isobutanol,ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol [propyleneglycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g.,pentane, hexane, heptane, octane, nonane, decane, undecane, anddodecane), a cycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane,and cyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene);an amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine,serine, and threonine); a gas (e.g., methane, hydrogen (H₂), carbondioxide (CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g.,acetone); an organic acid (e.g., acetic acid, acetonic acid, adipicacid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formicacid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid,glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid,malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid,succinic acid, and xylonic acid); and polyketide. The fermentationproduct can also be protein as a high value product.

In one aspect, the fermentation product is an alcohol. It will beunderstood that the term “alcohol” encompasses a substance that containsone or more hydroxyl moieties. The alcohol can be, but is not limitedto, n-butanol, isobutanol, ethanol, methanol, arabinitol, butanediol,ethylene glycol, glycerin, glycerol, 1,3-propanediol, sorbitol, xylitol.See, for example, Gong et al., 1999, Ethanol production from renewableresources, in Advances in Biochemical Engineering/Biotechnology,Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65:207-241; Silveira and Jonas, 2002, Appl. Microbiol. Biotechnol. 59:400-408; Nigam and Singh, 1995, Process Biochemistry 30(2): 117-124;Ezeji et al., 2003, World Journal of Microbiology and Biotechnology19(6): 595-603.

In another aspect, the fermentation product is an alkane. The alkane maybe an unbranched or a branched alkane. The alkane can be, but is notlimited to, pentane, hexane, heptane, octane, nonane, decane, undecane,or dodecane.

In another aspect, the fermentation product is a cycloalkane. Thecycloalkane can be, but is not limited to, cyclopentane, cyclohexane,cycloheptane, or cyclooctane.

In another aspect, the fermentation product is an alkene. The alkene maybe an unbranched or a branched alkene. The alkene can be, but is notlimited to, pentene, hexene, heptene, or octene.

In another aspect, the fermentation product is an amino acid. Theorganic acid can be, but is not limited to, aspartic acid, glutamicacid, glycine, lysine, serine, or threonine. See, for example, Richardand Margaritis, 2004, Biotechnology and Bioengineering 87(4): 501-515.

In another aspect, the fermentation product is a gas. The gas can be,but is not limited to, methane, H₂, CO₂, or CO. See, for example,Kataoka et al., 1997, Water Science and Technology 36(6-7): 41-47; andGunaseelan, 1997, Biomass and Bioenergy 13(1-2): 83-114.

In another aspect, the fermentation product is isoprene.

In another aspect, the fermentation product is a ketone. It will beunderstood that the term “ketone” encompasses a substance that containsone or more ketone moieties. The ketone can be, but is not limited to,acetone.

In another aspect, the fermentation product is an organic acid. Theorganic acid can be, but is not limited to, acetic acid, acetonic acid,adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid,formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronicacid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lacticacid, malic acid, malonic acid, oxalic acid, propionic acid, succinicacid, or xylonic acid. See, for example, Chen and Lee, 1997, Appl.Biochem. Biotechnol. 63-65: 435-448.

In another aspect, the fermentation product is polyketide.

Recovery. The fermentation product(s) can be optionally recovered fromthe fermentation medium using any method known in the art including, butnot limited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Signal Peptides

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to19 of SEQ ID NO: 2, amino acids 1 to 19 of SEQ ID NO: 4, amino acids 1to 19 of SEQ ID NO: 6, or amino acids 1 to 16 of SEQ ID NO: 8. Thepolynucleotide may further comprise a gene encoding a protein, which isoperably linked to the signal peptide. The protein is preferably foreignto the signal peptide. In one aspect, the polynucleotide encoding thesignal peptide is nucleotides 1 to 57 of SEQ ID NO: 1, nucleotides 1 to57 of SEQ ID NO: 3, nucleotides 1 to 57 of SEQ ID NO: 5, or nucleotides1 to 48 of SEQ ID NO: 7.

The present invention also relates to nucleic acid constructs,expression vectors and recombinant host cells comprising suchpolynucleotides.

The present invention also relates to methods of producing a protein,comprising (a) cultivating a recombinant host cell comprising suchpolynucleotide; and optionally (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andpolypeptides. The term “protein” also encompasses two or morepolypeptides combined to form the encoded product. The proteins alsoinclude hybrid polypeptides and fused polypeptides.

Preferably, the protein is a hormone, enzyme, receptor or portionthereof, antibody or portion thereof, or reporter. For example, theprotein may be a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an alpha-galactosidase, alpha-glucosidase,aminopeptidase, amylase, beta-galactosidase, beta-glucosidase,beta-xylosidase, carbohydrase, carboxypeptidase, catalase,cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, endoglucanase, esterase,glucoamylase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Strains

Lentinus similis was used as the source of genomic DNA encoding GH61polypeptides. The strain was isolated from a Chinese environmentalsample.

Bulgaria inquinans 046TR was used as the source of genomic DNA encodinga GH61 polypeptide. The strain is a natural isolate from a sampleobtained in Trorød Denmark. Bulgaria inquinans 046TR was propagated onPDA plates at 20° C.

Aspergillus oryzae MT3568 strain was used for expression of the GH61polypeptides. A. oryzae MT3568 is an amdS (acetamidase) disrupted genederivative of Aspergillus oryzae JaL355 (WO 2002/40694) in which pyrGauxotrophy was restored by disrupting the A. oryzae acetamidase (amdS)gene.

Media and Solutions

COVE sucrose plates were composed of 342 g of sucrose, 20 g of agarpowder, 20 ml of COVE salt solution, and deionized water to 1 liter. Themedium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). Themedium was cooled to 60° C. and then acetamide to 10 mM, CsCl to 15 mM,and TRITON® X-100 (50 μl/500 ml) were added.

COVE top agarose was composed of 342.3 g of sucrose, 20 ml of COVE saltsolution, 10 mM acetamide, 15 mM CsCl, 6 g of SEAKEM® GTG® agarose(Lonza Group Ltd., Basel, Switzerland), and deionized water to 1 liter.

COVE-2 plates for isolation were composed of 30 g of sucrose, 20 ml ofCOVE salt solution, 10 mM acetamide, 30 g of Noble agar, and deionizedwater to 1 liter.

COVE salt solution was composed of 26 g of MgSO₄.7H₂O, 26 g of KCl, 26 gof KH₂PO₄, 50 ml of COVE trace metals solution, and deionized water to 1liter.

COVE trace metals solution was composed of 0.04 g of Na₂13₄O₇.10H₂O, 0.4g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄. H₂O, 0.8 g ofNa₂MoO₄.2H₂O, 10 g of ZnSO₄.7H₂O, and deionized water to 1 liter.

Dap-4C medium was composed of 20 g of dextrose, 10 g of maltose, 11 g ofMgSO₄.7H₂O, 1 g of KH₂PO₄, 2 g of citric acid, 5.2 g of K₃PO₄. H₂O, 0.5g of yeast extract (Difco), 1 ml of antifoam, 0.5 ml of KU6 trace metalssolution, 2.5 g of CaCO₃, and deionized water to 1 liter. The medium wassterilized by autoclaving at 15 psi for 15 minutes (BacteriologicalAnalytical Manual, 8th Edition, Revision A, 1998). Before use, 3.5 ml ofsterile 50% (NH₄)₂HPO₄ and 5 ml of sterile 20% lactic acid were addedper 150 ml.

KU6 trace metals solution was composed of 0.13 g of NiCl₂, 2.5 g ofCuSO₄.5H₂O, 13.9 g of FeSO₄.7H₂O, 8.45 g of MnSO₄.H₂O, 6.8 g of ZnCl₂, 3g of citric acid, and deionized water to 1 liter.

LB medium was composed of 10 g of Bacto-Tryptone, 5 g of yeast extract,and 10 g of sodium chloride, and deionized water to 1 liter.

LB agar plates were composed of 10 g of Bacto-Tryptone, 5 g of yeastextract, 10 g of sodium chloride, 15 g of Bacto-agar, and deionizedwater to 1 liter.

PDA plates were composed of potato infusion made by boiling 300 g ofsliced (washed but unpeeled) potatoes in water for 30 minutes and thendecanting or straining the broth through cheesecloth. Distilled waterwas then added until the total volume of the suspension was one liter.Then 20 g of dextrose and 20 g of agar powder were added. The medium wassterilized by autoclaving at 15 psi for 15 minutes (BacteriologicalAnalytical Manual, 8th Edition, Revision A, 1998).

YP+2% glucose medium was composed of 1% yeast extract, 2% peptone, and2% glucose in deionized water.

YP+2% maltodextrin medium was composed of 1% yeast extract, 2% peptone,and 2% maltodextrin in deionized water.

60% PEG solution was composed of 60% (w/v) polyethyleneglycol (PEG)4000, 10 mM CaCl₂, and 10 mM Tris-HCl pH 7.5 in deionized water. Thesolution was filtered using a 0.22 μm PES membrane filter (MilliporeCorp., Billerica, Mass., USA) for sterilization. After filtersterilization, the PEG 60% was stored in aliquots at −20 C until use.

Example 1: Lentinus Similis Genomic DNA Extraction and Generation of DNASequence Information

Lentinus similis was grown on PDA plates at 26° C. for 7 days. Sporesharvested from the PDA plates were inoculated into 25 ml of YP+2%glucose medium in a baffled shake flask and incubated at 26° C. for 4days with agitation at 200 rpm.

Genomic DNA was isolated using a DNEASY® Plant Maxi Kit (QIAGEN Danmark,Copenhagen, Denmark) according to the following protocol. The fungalmaterial from the above culture was harvested by centrifugation at14,000×g for 2 minutes. The supernatant was removed and 0.5 g of thepellet was frozen in liquid nitrogen with quartz sand and ground to afine powder in a pre-chilled mortar. The powder was transferred to a 15ml centrifuge tube followed by 5 ml of AP1 buffer (QIAGEN Danmark,Copenhagen, Denmark) preheated to 65° C. and 10 μl of RNase A stocksolution (100 mg/ml). The mix was then vigorously vortexed. Afterincubation for 10 minutes at 65° C. with regular inverting of the tube,1.8 ml of AP2 buffer (QIAGEN Danmark, Copenhagen, Denmark) were added tothe lysate by gentle mixing followed by incubation on ice for 10minutes. The lysate was then centrifuged at 3000×g for 5 minutes at roomtemperature. The supernatant was decanted into a QIASHREDDER® Maxi SpinColumn (QIAGEN Danmark, Copenhagen, Denmark) placed in a 50 mlcollection tube and centrifuged at 3000×g for 5 minutes at roomtemperature. The flow-through was transferred to a new 50 ml tube and1.5 volumes of AP3/E buffer (QIAGEN Danmark, Copenhagen, Denmark) wereadded followed by vortexing. A total of 15 ml of the sample wastransferred into a DNEASY® Maxi Spin Column (QIAGEN Danmark, Copenhagen,Denmark) placed in a 50 ml collection tube and centrifuged at 3000×g for5 minutes at room temperature. The flow-through was discarded and 12 mlof AW buffer (QIAGEN Danmark, Copenhagen, Denmark) were added to theDNEASY® Maxi Spin Column placed in a 50 ml collection tube andcentrifuged at 3000×g for 10 minutes at room temperature. Afterdiscarding the flow-through, the centrifugation was repeated to disposeof the remaining alcohol. The DNEASY® Maxi spin column was transferredto a new 50 ml tube and 0.5 ml of AE buffer (QIAGEN Danmark, Copenhagen,Denmark) preheated to 70° C. was added. After incubation at roomtemperature for 5 minutes, the sample was eluted by centrifugation at3000×g for 5 minutes at room temperature. Elution was repeated with anadditional 0.5 ml of AE buffer and the eluates were combined. Theconcentration of the harvested DNA was measured by UV at 260 nm.

Example 2: Generation of Lentinus Similis DNA Sequence Information

Two μg of the Lentinus similis genomic DNA (Example 1) was subjected topartial shotgun genome sequencing, using a service commerciallyavailable at FASTERIS SA, Switzerland. The genome sequence was analyzedfor protein sequences that have GH61 glycosyl hydrolase domains(according to the CAZY definition above). Three genes and correspondingprotein sequences were identified from the sequence information (SEQ IDNO: 1, SEQ ID NO: 3, and SEQ ID NO: 5).

Example 3: Construction of Aspergillus oryzae Expression VectorsContaining Lentinus Similis Genomic Sequences Each Encoding a GH61Polypeptide

Two synthetic oligonucleotide primers, shown below, were designed foreach of the three GH61 polypeptide genes to PCR amplify the Lentinussimilis GH61 coding sequences from the genomic DNA prepared inExample 1. An IN-FUSION™ PCR Cloning Kit (BD Biosciences, Palo Alto,Calif., USA) was used to clone the fragments directly into theexpression vector pDau109 (WO 2005/042735).

For amplification of the GH61 polypeptide coding sequence of SEQ ID NO:1, the following primers were used:

Primer F-P247JE: (SEQ ID NO: 9)5′-ACACAACTGGGGATCCACCATGCGCGGATTCGCTTCTCT-3′ Primer R-P247JE:(SEQ ID NO: 10) 5′-CCCTCTAGATCTCGAG GCTGAAGCTCCCTATCACGAAGTA-3′

For amplification of the GH61 polypeptide coding sequence of SEQ ID NO.3, the following primers were used:

Primer F-P247J6: (SEQ ID NO: 11)5′-ACACAACTGGGGATCCACCATGAAGCTCTCAGCTCTCGTAGCT-3′ Primer R-P247J6:(SEQ ID NO. 12) 5′-CCCTCTAGATCTCGAG TCAGCGTGGCACATGGGTT-3′

For amplification of the GH61 polypeptide coding sequence of SEQ ID NO:5, the following primers were used:

Primer F-P247JK: (SEQ ID NO: 13)5′-ACACAACTGGGGATCCACCATGAAGTACTCCATCCTCGGGCT-3′ Primer R-P247JK:(SEQ ID NO: 14) 5′-CCCTCTAGATCTCGAG CCTTGTCGAGCGACTCTATCCA-3′Bold letters represent gene sequences. The underlined sequences arehomologous to the insertion sites of pDau109.

A PHUSION® High-Fidelity PCR Kit (Finnzymes Oy, Espoo, Finland) was usedfor the PCR amplification. The PCR reactions were composed of 5 μl of5×HF buffer (Finnzymes Oy, Espoo, Finland), 0.5 μl of dNTPs (10 mM), 0.5μl of PHUSION® DNA polymerase (0.2 units/μl) (Finnzymes Oy, Espoo,Finland), 5 μM of each primer, 0.5 μl of L. similis genomic DNA (100ng/μl), and 16.5 μl of deionized water in a total volume of 25 μl. ThePCR reactions were performed using a PTC-200 DNA Engine (MJ ResearchInc., Waltham, Mass., USA) programmed for 1 cycle at 95° C. for 2minutes; 35 cycles each at 98° C. for 10 seconds, 60° C. for 30 seconds,and 72° C. for 2 minutes; and 1 cycle at 72° C. for 10 minutes. Thesamples were then held at 12° C. until removed from the PCR machine.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing 40 mM Tris base, 20 mM sodium acetate, 1 mM disodium EDTA (TAE)buffer where product bands of approximately 1200-1400 bp were excisedfrom the gels and purified using an ILLUSTRA® GFX® PCR DNA and Gel BandPurification Kit (GE Healthcare Life Sciences, Brondby, Denmark). Thefragments were then cloned into Barn HI and Xho I digested pDau109 usingan IN-FUSION™ Cloning Kit resulting in plasmids pP247JE, pP247J6, andpP247JK for SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively.Cloning of the genes into Barn HI-Xho I digested pDau109 resulted intranscription of the Lentinus similis GH61 genes under the control of aNA2-tpi double promoter. NA2-tpi is a modified promoter from the geneencoding Aspergillus niger neutral alpha-amylase in which theuntranslated leader has been replaced by an untranslated leader from thegene encoding Aspergillus nidulans triose phosphate isomerase.

The cloning protocol was performed according to the IN-FUSION™ CloningKit instructions generating three GH61 constructs. The treated plasmidsand inserts were transformed into One Shot® TOP10F′ Chemically CompetentE. coli cells (Invitrogen, Carlsbad, Calif., USA) according to themanufacturer's protocol and spread onto LB plates supplemented with 0.1mg of ampicillin per ml. After incubation at 37° C. overnight, colonieswere observed growing under selection on the LB ampicillin plates.Colonies of each transformation were cultivated in LB mediumsupplemented with 0.1 mg of ampicillin per ml and plasmids were isolatedusing a QIAPREP® Spin Miniprep Kit (QIAGEN Inc., Valencia, Calif., USA).

Isolated plasmids were sequenced with vector primers and gene specificprimers in order to determine representative plasmid expression clonesthat were free of PCR errors, and plasmids without errors were selectedfor expression of the GH61 polypeptides.

Example 4: Characterization of the Lentinus Similis Genomic SequencesEncoding GH61 Polypeptides

DNA sequencing of the Lentinus similis GH61 genomic clones was performedusing an Applied Biosystems Model 3700 Automated DNA Sequencer andversion 3.1 BIG-DYE™ terminator chemistry (Applied Biosystems, Inc.,Foster City, Calif., USA) and primer walking strategy. Nucleotidesequence data were scrutinized for quality and all sequences werecompared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA). The sequences obtainedwere identical to the sequences from the genome sequencing (see Example1).

The nucleotide sequence and deduced amino acid sequence of the Lentinussimilis P247JE gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2,respectively. The coding sequence of P247JE is 1461 bp including thestop codon and is interrupted by 9 introns of 59 bp (nucleotides 73 to131), 56 bp (nucleotides 198 to 253), 56 bp (nucleotides 381 to 436), 52bp (nucleotides 701 to 752), 54 bp (nucleotides 794 to 847), 57 bp(nucleotides 927 to 983), 53 bp (nucleotides 1044 to 1096), 62 bp(nucleotides 1263 to 1324), and 61 bp (nucleotides 1383 to 1443). Theencoded predicted protein is 316 amino acids. Using the SignalP 3.0program (Bendtsen et al., 2004, supra), a signal peptide of 19 residueswas predicted. The SignalP prediction is in accord with the necessityfor having a histidine reside at the N-terminus in order for propermetal binding and hence protein function to occur (See Harris et al.,2010, Biochemistry 49: 3305, and Quinlan et al., 2011, Proc. Natl. Acad.Sci. USA 108: 15079). The GH61 catalytic domain and CBM domain werepredicted to be amino acids 20 to 245 and amino acids 284 to 316,respectively, by aligning the amino acid sequence of the full-lengthprotein using BLAST to all CAZY-defined subfamily module subsequences(Cantarel et al., 2009, Nucleic Acids Res. 37: D233-238), where thesingle most significant alignment within a subfamily was used to predictthe location of GH61 catalytic and CBM domains. The predicted matureprotein contains 297 amino acids.

The nucleotide sequence and deduced amino acid sequence of the Lentinussimilis P247J6 gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4,respectively. The coding sequence of P247J6 is 988 bp including the stopcodon and is interrupted by 5 introns of 63 bp (nucleotides 62 to 124),55 bp (nucleotides 353 to 407), 58 bp (nucleotides 614 to 671), 54 bp(nucleotides 828 to 881), and 62 bp (nucleotides 909 to 970). Theencoded predicted protein is 231 amino acids. Using the SignalP 3.0program (Bendtsen et al., 2004, supra), a signal peptide of 19 residueswas predicted. The SignalP prediction is in accord with the necessityfor having a histidine reside at the N-terminus in order for propermetal binding and hence protein function to occur (See Harris et al.,2010, supra, and Quinlan et al., 2011, supra). The predicted maturepolypeptide contains 212 amino acids.

The nucleotide sequence and deduced amino acid sequence of the Lentinussimilis P247JK gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6,respectively. The coding sequence of P247JK is 1072 bp including thestop codon and is interrupted by 5 introns of 76 bp (nucleotides 197 to272), 55 bp (nucleotides 337 to 391), 50 bp (nucleotides 396 to 445), 63bp (nucleotides 660 to 722), and 63 bp (nucleotides 785 to 847). Theencoded predicted protein is 254 amino acids. Using the SignalP 3.0program (Bendtsen et al., 2004, supra), a signal peptide of 19 residueswas predicted. The SignalP prediction is in accord with the necessityfor having a histidine reside at the N-terminus in order for propermetal binding and hence protein function to occur (See Harris et al.,2010, supra, and Quinlan et al., 2011, supra). The predicted maturepolypeptide contains 235 amino acids.

Example 5: Expression of Lentinus Similis GH61 Coding Sequences inAspergillus Oryzae M13568

Error-free clones comprising the P247JE GH61 polypeptide coding sequenceof SEQ ID NO: 1, the P247J6 GH61 polypeptide coding sequence of SEQ IDNO: 3, and the P247JK GH61 polypeptide coding sequence of SEQ ID NO: 5were expressed in Aspergillus oryzae MT3568. Plasmid DNA was isolated asdescribed in Example 3 and transformed into Aspergillus oryzae MT3568.A. oryzae MT3568 protoplasts were prepared according to the method ofEuropean Patent EP0238023, pages 14-15. Transformants resulting from thetransformation of A. oryzae MT3568 with each of the three plasmids wereinoculated into separate wells of a 96 microtiter deep well plate (NuncA/S, Roskilde, Denmark) with each well containing 750 μl of YP+2%glucose medium or 750 μl of YP+2% maltodextrin medium. The plate wascovered with Nunc pre scored vinyl sealing tape (Thermo FisherScientific, Roskilde, Denmark) and incubated at 26° C. stationary for 4days. The transformants were also streaked onto COVE sucrose platescontaining 10 mM acetamide, 15 mM CsCl, and TRITON® X-100 (50 μl/500ml). The plates were incubated at 37° C. and this selection procedurewas repeated in order to stabilize the transformants.

Several Aspergillus oryzae transformants produced the P247JE GH61polypeptide of SEQ ID NO: 2, the P247J6 GH61 polypeptide of SEQ ID NO:4, or the P247JK GH61 polypeptide of SEQ ID NO: 6 as judged by SDS PAGEanalysis.

Example 6: Purification of the Lentinus Similis P247JE Polypeptide

An Aspergillus oryzae transformant producing the recombinant P247JE GH61polypeptide (Example 5) was inoculated in 2 liters of Dap-4C medium andincubated at 30° C. for 4 days. Mycelia were removed by filtration andthe broth collected for purification of protein.

The broth was sterile filtered, washed, and concentrated byultrafiltration first using a 0.1 m² polysulphone (PES) 10 kDa cutoffmembrane on a Sartojet pump (Sartorius Stedim, Goettingen, Germany) andthen using VIVASPIN® 20 (10 KDa MWCO) spin concentrators (SartoriusStedium Biotech, Goettingen, Germany). The enzyme was purified using a26/60 SUPERDEX™ 75 column (GE Healthcare Bio-Sciences AB, Uppsala,Sweden) with isocratic elution using 20 mM2-(N-morpholino)ethanesulfonic acid (MES)-125 mM sodium chloride pH 6.0.The injection volume was limited to 5 ml per run.

Fractions containing GH61 polypeptide were pooled based on A280absorption and concentrated by ultrafiltration using VIVASPIN® 20 (10KDa MWCO) spin concentrators. Protein concentration was determined byA280 absorption.

Example 7: Purification of the Lentinus Similis P247J6 GH61 Polypeptide

An Aspergillus oryzae transformant producing the recombinant P247J6 GH61polypeptide (Example 5) was inoculated into 2 liters of Dap-4C mediumand incubated at 30° C. for 4 days. Mycelia were removed by filtrationand the broth collected for purification of protein.

Ammonium sulfate (AMS) was added to the sterile filtered broth to 1 Mand the pH adjusted to 7.5. The broth was applied to a 50/15 ButylToyopearl column (Tosoh Biosciences, Stuttgart, Germany) equilibratedwith 25 mM trisaminomethane (Tris), 1.0 M AMS pH 7.5. The column waswashed in the same buffer and eluted with a gradient from 0 to 25 mMTris pH 7.5. Fractions containing GH61 polypeptide were pooled based onA₂₈₀ absorption and washed with 25 mM Tris pH 7.5 by ultrafiltrationusing VIVASPIN® 20 (10 KDa MWCO) spin concentrators to a conductivity of2 mSi/cm. The pH was adjusted to 9.0 and applied to a 26/20 Q SEPHAROSE®column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) equilibratedwith 20 mM Tris pH 9.0. The column was washed in the same buffer and theenzyme eluted with a gradient from 0 to 0.5 M sodium chloride in 20 mMTris pH 9.0. Fractions containing GH61 polypeptide were pooled based onA280 absorption and concentrated by ultrafiltration using VIVASPIN® 20(10 KDa MWCO) spin concentrators. The enzyme was further purified on a26/60 SUPERDEX™ 75 column with isocratic elution using 20 mM2-(N-morpholino)ethanesulfonic acid (MES)-125 mM sodium chloride pH 6.0.The injection volume was limited to 5 ml per run. Protein concentrationwas determined by A280 absorption.

Example 8: Purification of the Lentinus Similis P247JK GH61 Polypeptide

An Aspergillus oryzae transformant producing the recombinant P247JK GH61polypeptide (Example 5) was inoculated in 2 liters of Dap-4C medium andincubated at 30° C. for 4 days. Mycelia were removed by filtration andthe broth collected for purification of protein.

Ammonium sulfate (AMS) was added to the sterile filtered broth to 1 Mand the pH adjusted to 7.5. The broth was applied to a 50/15 ButylToyopearl column (Tosoh Biosciences, Stuttgart, Germany) equilibratedwith 25 mM Tris, 1.0 AMS pH 7.5. The column was washed in the samebuffer and eluted with a gradient from 0 to 25 mM Tris pH 7.5. Fractionscontaining GH61 polypeptide were pooled on A280 absorption and washedwith 25 mM Tris pH 7.5 by ultrafiltration using VIVASPIN® 20 (10 KDaMWCO) spin concentrators to a conductivity of 4.1 mSi/cm. The pH wasadjusted to 8.0 and applied to a 26/2 Q SEPHAROSE® column equilibratedwith 20 mM Tris pH 8.0. The column was washed in the same buffer and theenzyme eluted with a gradient from 0 to 0.5 M sodium chloride. Fractionscontaining GH61 polypeptide based on A280 absorption were pooled andconcentrated by ultrafiltration using VIVASPIN® 20 (10 KDa MWCO) spinconcentrators. The enzyme was further purified on a 26/60 SUPERDEX™ 75column with isocratic elution using 20 mM 2-(N-morpholino)ethanesulfonicacid (MES)-125 mM sodium chloride pH 6.0. The injection volume waslimited to 5 ml per run. Fractions containing GH61 polypeptide werepooled based on A280 absorption. Protein concentration determined byA280 absorption.

Example 9: Preparation of Trichoderma reesei GH5 Endoglucanase II

The Trichoderma reesei GH5 endoglucanase II (SEQ ID NO: 15 [DNAsequence] and SEQ ID NO: 16 [deduced amino acid sequence]) was preparedrecombinantly according to WO 2011/057140 using Aspergillus oryzae as ahost. The filtered broth of the T. reesei endoglucanase II was desaltedand buffer-exchanged into 20 mM Tris pH 8.0 using a tangential flowconcentrator (Pall Filtron, Northborough, Mass., USA) equipped with a 10kDa polyethersulfone membrane (Pall Filtron, Northborough, Mass., USA).The protein concentration was determined using a Microplate BCA™ ProteinAssay Kit (Thermo Fischer Scientific, Waltham, Mass., USA) in whichbovine serum albumin was used as a protein standard.

Example 10: Preparation of Aspergillus fumigatus Cel3A Beta-Glucosidase

The Aspergillus fumigatus NN055679 Cel3A beta-glucosidase. (SEQ ID NO:17 [DNA sequence] and SEQ ID NO: 18 [deduced amino acid sequence]) wasrecombinantly prepared according to WO 2005/047499 using Aspergillusoryzae as a host. The filtered broth was adjusted to pH 8.0 with 20%sodium acetate, which made the solution turbid. To remove the turbidity,the solution was centrifuged at 20,000×g for 20 minutes, and thesupernatant was filtered through a 0.2 μm filtration unit (Nalgene,Rochester, N.Y., USA). The filtrate was diluted with deionized water toreach the same conductivity as 50 mM Tris-HCl pH 8.0. The adjustedenzyme solution was applied to a Q SEPHAROSE® Fast Flow column (GEHealthcare, Piscataway, N.J., USA) equilibrated in 50 mM Tris-HCl pH 8.0and eluted with a linear 0 to 500 mM sodium chloride gradient. Fractionswere pooled and treated with 1% (w/v) activated charcoal to remove colorfrom the beta-glucosidase pool. The charcoal was removed by filtrationof the suspension through a 0.2 μm filtration unit. The filtrate wasadjusted to pH 5.0 with 20% acetic acid and diluted 10 times withdeionized water. The adjusted filtrate was applied to a SP SEPHAROSE®Fast Flow column (GE Healthcare, Piscataway, N.J., USA) equilibrated in10 mM succinic acid pH 5.0 and eluted with a linear 0 to 500 mM sodiumchloride gradient. Fractions were collected and analyzed forbeta-glucosidase activity using p-nitrophenyl-beta-D-glucopyranoside assubstrate. A p-nitrophenyl-beta-D-glucopyranoside stock solution wasprepared by dissolving 50 mg of the substrate in 1.0 ml of DMSO. Justbefore use a substrate solution was prepared by mixing 100 μl of thestock solution with 4900 μl of 100 mM succinic acid, 100 mM HEPES, 100mM CHES, 100 mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% TRITON® X-100, pH5.0 (assay buffer). A 200 μl volume of the substrate solution wasdispensed into a tube and placed on ice followed by 20 μl of enzymesample (diluted in 0.01% TRITON® X-100). The assay was initiated bytransferring the tube to a thermomixer, which was set to an assaytemperature of 37° C. The tube was incubated for 15 minutes on thethermomixer at its highest shaking rate (1400 rpm). The assay wasstopped by transferring the tube back to the ice bath and adding 600 μlof a Stop solution (500 mM H₃BO₃/NaOH pH 9.7). Then the tube was mixedand allowed to reach room temperature. A 200 μl of supernatant wastransferred to a microtiter plate and the absorbance at 405 nm was readas a measure of beta-glucosidase activity. A buffer control was includedin the assay (instead of enzyme). Fractions with beta-glucosidaseactivity were further analyzed by SDS-PAGE using CRITERION™ 8-16%Stain-Free Tris-HCl gel (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA) Fractions, where only one band was seen on a Coomassie blue stainedSDS-PAGE gel, were pooled as the purified product. The proteinconcentration was determined using a Microplate BCA™ Protein Assay Kitin which bovine serum albumin was used as a protein standard.

Example 11: Microcrystalline Cellulose Hydrolysis Assay

A 5% microcrystalline cellulose slurry was prepared by addition of 2.5 gof microcrystalline cellulose (AVICEL® PH101; Sigma-Aldrich, St. Louis,Mo., USA) to a graduated 50 ml screw-cap conical tube followed byapproximately 40 ml of double-distilled water. The conical tube was thenmixed thoroughly by shaking/vortexing, and adjusted to 50 ml total withdouble-distilled water and mixed again. The contents of the tube werethen quickly transferred to a 100 ml beaker and stirred rapidly with amagnetic stirrer.

The hydrolysis of microcrystalline cellulose was conducted using a 2.2ml deep-well plate (Axygen, Union City, Calif., USA) in a total reactionvolume of 1.0 ml. The hydrolysis was performed with 25 mg of themicrocrystalline cellulose slurry (containing 100% cellulose) per ml ofreaction. A 500 μl aliquot of the 5% microcrystalline cellulose slurrywas pipetted into each well of the 2.2 ml deep-well plate using a 1000μl micropipette with a wide aperture tip (end of tip cut off about 2 mmfrom the base). Each reaction was performed with and without theaddition of catechol. In reactions not containing catechol, 200 μl ofdouble-distilled water were added to each well. Then 100 μl of 500 mMammonium acetate pH 5.0 containing 100 μM copper sulfate or 100 μl of500 mM ammonium acetate pH 8.0 containing 100 μM copper sulfate wereadded to each well. An enzyme composition consisting of Trichodermareesei GH5 endoglucanase II (loaded at 2 mg protein per g cellulose) andAspergillus fumigatus GH3A beta-glucosidase (loaded at 2 mg protein perg cellulose) was prepared and then added simultaneously to each well ina volume of 100 μl. A solution containing the GH61 polypeptide (loadedat 5 mg protein per g cellulose) was then added to each well in a volumeof 100 μl for a final volume of 1 ml in each reaction not containingcatechol. In the reactions containing catechol, 200 μl of 100 mMcatechol were added to each of the appropriate wells for a final volumeof 1 ml and a final catechol concentration of 20 mM. The plate was thensealed using an ALPS-300™ plate heat sealer (Abgene, Epsom, UnitedKingdom), mixed thoroughly, and incubated at 50° C. for 72 hours. Allexperiments reported were performed in triplicate.

Following hydrolysis, samples were filtered using a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA) and filtratesanalyzed for glucose content as described below. When not usedimmediately, filtered aliquots were frozen at −20° C. The sugarconcentrations of the samples were measured using a 4.6×250 mm AMINEX®HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) byelution with 0.05% w/w benzoic acid-0.005 M H₂SO₄ at 65° C. at a flowrate of 0.6 ml per minute, and quantitation by integration of theglucose signal from refractive index detection (CHEMSTATION®, AGILENT®1100 HPLC, Agilent Technologies, Santa Clara, Calif., USA) calibrated bypure glucose samples.

All HPLC data processing was performed using MICROSOFT EXCEL™ software(Microsoft, Richland, Wash., USA). The resultant glucose was used forcomparison of each reaction. Triplicate data points were averaged andstandard deviation was calculated.

Example 12: Effect of the Lentinus Similis GH61 Polypeptides on theHydrolysis of Microcrystalline Cellulose

The Lentinus similis P247JE and P247J6 GH61 polypeptides were eachevaluated for the ability to enhance the hydrolysis of microcrystallinecellulose in the presence of Trichoderma reesei GH5 endoglucanase II(loaded at 2 mg protein per g cellulose) and Aspergillus fumigatus GH3Abeta-glucosidase (loaded at 2 mg protein per g cellulose) with andwithout the addition of 20 mM catechol at 50° C. The Lentinus similisGH61 polypeptides were added separately at 5 mg protein per g cellulose.The composition of T. reesei GH5 endoglucanase II (loaded at 2 mgprotein per g cellulose) and A. fumigatus GH3A beta-glucosidase (loadedat 2 mg protein per g cellulose) with and without 20 mM catechol wasalso run as controls without added GH61 polypeptide.

The assay was performed as described in Example 11. The 1 ml reactionswith microcrystalline cellulose were conducted for 72 hours in 50 mMammonium acetate pH 5.0 containing 10 μM copper sulfate or 50 mMammonium acetate pH 8.0 containing 10 μM copper sulfate. All reactionswere performed in triplicate and involved single mixing at the beginningof hydrolysis.

As shown in FIGS. 1 and 2, hydrolysis of the microcrystalline celluloseby the composition of T. reesei GH5 endoglucanase II and A. fumigatusGH3A beta-glucosidase without catechol produced similar results as thatobtained with either L. similis GH61 polypeptide added to thecomposition of T. reesei GH5 endoglucanase II and A. fumigatus GH3Abeta-glucosidase without catechol. The addition of L. similis GH61polypeptide to the composition of T. reesei GH5 endoglucanase II and A.fumigatus GH3A beta-glucosidase without catechol did not improvehydrolysis of the microcrystalline cellulose. However, as shown in FIGS.1 and 2, the addition of either L. similis GH61 polypeptide to thecomposition of T. reesei GH5 endoglucanase II and A. fumigatus GH3Abeta-glucosidase with 20 mM catechol resulted in a higher degree ofglucose production (shown in g/L) compared to the addition of either L.similis GH61 polypeptide to the composition of T. reesei GH5endoglucanase II and A. fumigatus GH3A beta-glucosidase without addedcatechol and compared to the composition of T. reesei GH5 endoglucanaseII and A. fumigatus GH3A beta-glucosidase without GH61 polypeptide andwith or without added catechol. As shown in FIG. 1, the resultsdemonstrated a 1.82-fold improvement (or 82% increase) or 1.15-foldimprovement (or 15% increase) in hydrolysis of the microcrystallinecellulose by L. similis GH61 polypeptide addition (L. similis GH61polypeptide P247JE and L. similis GH61 polypeptide P247JE, respectively)to the composition of T. reesei GH5 endoglucanase II and A. fumigatusGH3A beta-glucosidase with catechol compared to without catechol at pH5.0. As shown in FIG. 2, the results demonstrated a 1.29-foldimprovement (or 29% increase) in hydrolysis of the microcrystallinecellulose by L. similis P247JE GH61 polypeptide addition to thecomposition of T. reesei GH5 endoglucanase II and A. fumigatus GH3Abeta-glucosidase with catechol compared to without catechol at pH 8.0.

Example 13: Preparation of Phosphoric Acid Swollen Cellulose (PASC)

A 1% phosphoric acid swollen cellulose (PASO) slurry was prepared frommicrocrystalline cellulose (AVICEL® PH101, Sigma-Aldrich, St. Louis,Mo., USA) using the protocol described by Zhang et al., 2006,Biomacromolecules 7: 644-648.

Example 14: Preparation of Copper(II) Preincubated GH61 Polypeptides

Lentinus similis GH61 polypeptides at 0.25 gram per liter were eachpreincubated with equal molar copper sulfate at 23° C. for 30 minutes,prior to addition to a PASO hydrolysis suspension.

Example 15: Evaluation of the Degradation of Phosphoric Acid SwollenCellulose by Lentinus Similis GH61 Polypeptides

The activity of Lentinus similis GH61 polypeptides on phosphoric acidswollen cellulose (PASO) was evaluated according to the proceduredescribed below.

The degradation of PASO was conducted using 2.0 ml deep-well plates(Axygen Scientific, Union City, Calif., USA) in a total reaction volumeof 1.0 ml. Each hydrolysis was performed with 5 mg of PASO per ml of 100mM sodium tartrate, Bis-Tris, glycylglycine (TBG) buffer at pH 5(Molecular Dimensions Inc., Apopka, Fla., USA) containing L. similisP247JE, P247JK, or P247JE GH61 polypeptide at 10 mg protein per gram ofcellulose, 1 mM calcium chloride, plus pyrogallol,4-hydroxy-5-methyl-3-furanone, cysteine, or ascorbate at 5 mM ascofactor for the GH61 polypeptide. The plate was then sealed using anALPS-300™ or ALPS-3000™ plate heat sealer (Abgene, Epsom, UnitedKingdom), mixed thoroughly, and incubated at 50° C. for 1 day in anIsotemp Plus incubator (Thermo Fisher Scientific Inc., Waltham, Mass.,USA). The final pH was measured after completion of the reactions. Allexperiments were performed at least in triplicate.

Following hydrolysis, samples were filtered using a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA). The filtrateswere reacted with Aspergillus oryzae GH3A beta-glucosidase at 50 mgprotein per liter at 50° C. for at least 24 hours. As controls, a 1-hourbeta-glucosidase reaction was compared to a 24-hour reaction, and thebeta-glucosidase reaction without pH adjustment of the GH61-PASCreaction was compared to beta-glucosidase reaction with the pH of theGH61-PASC reaction adjusted to 5. No significant differences wereobserved.

Reacted samples were analyzed for glucose and cellobiose content asdescribed below. When not used immediately, filtered aliquots werefrozen at −20° C. The sugar concentrations of samples, diluted toappropriate concentrations in 0.005 M H₂SO₄, were measured using a4.6×250 mm AMINEX® HPX-87H column by elution with 0.05% (w/w) benzoicacid-0.005 M H₂SO₄ at 65° C. at a flow rate of 0.6 ml per minute, andquantitated by integration of the glucose and cellobiose signals fromrefractive index detection calibrated by pure sugar samples. Theresultant glucose and cellobiose equivalents were used to calculate thepercentage of cellulose conversion for each reaction. Measured sugarconcentrations were adjusted for the appropriate dilution factor. Datawere processed using MICROSOFT EXCEL™ software.

Percent conversion was calculated based on the mass ratio of solubilizedglucosyl units to the initial mass of insoluble cellulose. Only glucoseand cellobiose were measured for soluble sugars, as cellodextrins longerthan cellobiose were present in negligible concentrations (due toenzymatic hydrolysis). The extent of total cellulose conversion wascalculated using the following equation:

${\%\mspace{14mu}{conversion}} = \frac{\left( {\left( {\lbrack{cellobiose}\rbrack\left( {{mg}/{ml}} \right) \times 1.053} \right) + {\left( {\lbrack{glucose}\rbrack\left( {{mg}/{ml}} \right)} \right)/1.111}} \right)}{\lbrack{cellulose}\rbrack\left( {{mg}/{ml}} \right)}$

The 1.111 and 1.053 factors for glucose and cellobiose, respectively,take into account the increase in mass when the glucosyl units incellulose (average molecular mass of 162 daltons) are converted toglucose (molecular mass of 180 daltons) or cellobiose glucosyl units(average molecular mass of 171 daltons).

The results are shown in FIGS. 3-5. FIGS. 3, 4, and 5 show the activityof the Lentinus similis P247JE, P247JK, and P247J6 GH61 polypeptide,respectively, in degrading phosphoric acid-swollen cellulose (PASC).

Example 16: Cloning of the P24NHZ GH61 Polypeptide Coding Sequence fromBulgaria Inquinans 046TR

The P24NHZ GH61 polypeptide coding sequence was cloned from Bulgariainquinans 046TR genomic DNA by PCR.

Bulgaria inquinans 046TR was cultivated in 100 ml of YP+2% glucosemedium in 1000 ml Erlenmeyer shake flasks for 5 days at 20° C. Myceliawere harvested from the flasks by filtration of the cultivation mediumthrough a Buchner vacuum funnel lined with MIRACLOTH® (EMD Millipore,Billerica, Mass., USA). Mycelia were frozen in liquid nitrogen andstored at −80C until further use. Genomic DNA was isolated using aDNEASY® Plant Maxi Kit according to the manufacturer's instructions.

Genomic sequence information was generated by Illumina HiSeq 2000equipment at Fasteris SA, Switzerland, Plan-les-Ouates, Switzerland.Five μg of the isolated Bulgaria inquinans genomic DNA were sent toFasteris for preparation and analysis and a 100 bp, paired end strategywas employed with a library insert size of 200-500 bp. One half of aHiSeq run was used for a total of 2×133,440,122 100 bp raw readsobtained. The reads were subsequently fractionated to 25% (leaving2×33,360,030 reads) followed by trimming (extracting longestsub-sequences having Phred-scores of 10 or more). These reads wereassembled using ldba version 0.18. Contigs shorter than 200 bp werediscarded, resulting in 29,644,592 bp with an N-50 of 16,380. Genes werecalled using GeneMark.hmm ES version 2.3a and identification of thecatalytic domain was made using “Glyco_hydro_61” Hidden Markov Modelprovided by Pfam. The P24NHZ GH61 polypeptide coding sequence for theentire coding region was cloned from Bulgaria inquinans genomic DNA byPCR using the primers shown below.

Primer KKSC0136-F: (SEQ ID NO: 19)5′-ACACAACTGGGGATCCACCATGTCCACTTTGTTGGGCCT-3′ Primer KKSC0136-R:(SEQ ID NO: 20) 5′-AGATCTCGAGAAGCTT ACTAAGCATCGCAGGTGTCGT-3′Bold letters represent Bulgaria inquinans P24NHZ GH61 polypeptide codingsequence. Restriction sites are underlined. The sequence to the left ofthe restriction sites is homologous to the insertion sites of pDau109.

The amplification reaction (40 μl) was composed of 25 μl of 2× IPROOF™HF Master Mix (Bio-Rad Laboratories, Inc., Hercules, Calif., USA), 1 μlof primer KKSC0136F (100 μM), 1 μl of primer KKSC0136R (100 μM), 1 μl ofBulgaria inquinans genomic DNA (100 ng/μl), and 22 μl of deionizedwater. The PCR reaction was incubated in a DYAD® Dual-Block ThermalCycler (MJ Research Inc., Waltham, Mass., USA) programmed for 1 cycle at98° C. for 30 seconds; 30 cycles each at 98° C. for 10 seconds, 55° C.for 20 seconds, and 72° C. for 30 seconds; and 1 cycle at 72° C. for 10minutes. Samples were cooled to 10° C. before removal and furtherprocessing.

Five μl of the PCR reaction were analyzed by 1% agarose gelelectrophoresis using TAE buffer where a major band of about 1.1 kb wasobserved. The remaining PCR reaction was purified directly using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

Two μg of plasmid pDau109 were digested with Barn HI and Hind III andthe digested plasmid was run on a 1% agarose gel using 50 mM Trisbase-50 mM boric acid-1 mM disodium EDTA (TBE) buffer in order to removethe stuffer fragment from the restricted plasmid. The bands werevisualized by the addition of SYBR® Safe DNA gel stain (LifeTechnologies Corporation, Grand Island, N.Y., USA) and use of a 470 nmwavelength transilluminator. The band corresponding to the restrictedplasmid was excised and purified using an ILLUSTRA™ GFX™ PCR DNA and GelBand Purification Kit. The plasmid was eluted into 10 mM Tris pH 8.0 andits concentration adjusted to 20 ng per μl. An IN-FUSION® PCR CloningKit was used to clone the 1170 bp PCR fragment into pDau109 digestedwith Barn HI and Hind III (20 ng). The IN-FUSION® total reaction volumewas 10 μl. The IN-FUSION® reaction was transformed into FUSION-BLUE™ E.coli cells (Clontech Laboratories, Inc., Mountain View, Calif., USA)according to the manufacturer's protocol and spread onto LB agar platessupplemented with 50 μg of ampicillin per ml. After incubation overnightat 37° C., transformant colonies were observed growing under selectionon the LB agar plates.

Several colonies were selected for analysis by colony PCR using theprimers described below. Four colonies were transferred from the LB agarplates with a yellow inoculation pin (Nunc A/S, Denmark) to new LB agarplates supplemented with 50 μg of ampicillin per ml and incubatedovernight at 37° C.

Primer 8653: (SEQ ID NO: 21) 5′-GCAAGGGATGCCATGCTTGG-3′ Primer 8654:(SEQ ID NO: 22) 5′-CATATAACCAATTGCCCTC-3′

Each of the four colonies were transferred directly into 200 μl PCRtubes composed of 6 μl of 2× HiFi REDDYMIX™ PCR Master Mix (ThermoFisherScientific, Rockford, Ill., USA), 0.5 μl of primer 8653 (10 μm/μl), 0.5μl of primer 8654 (10 μm/μl), and 5 μl of deionized water. Each colonyPCR was incubated in a DYAD® Dual-Block Thermal Cycler programmed for 1cycle at 94° C. for 60 seconds; and 30 cycles each at 94° C. for 30seconds, 55° C. for 30 seconds, 68° C. for 60 seconds, 68° C. for 10minutes, and 10° C. for 10 minutes.

Four μl of each completed PCR reaction were submitted to 1% agarose gelelectrophoresis using TAE buffer. All four E. coli transformants showeda PCR band of 1.1 kb. Plasmid DNA was isolated from each of the fourcolonies using a QIAPREP® Spin Miniprep Kit. The resulting plasmid DNAwas sequenced with primers 8653 and 8654 using an Applied BiosystemsModel 3730 Automated DNA Sequencer and version 3.1 BIG-DYE™ terminatorchemistry (Applied Biosystems, Inc., Foster City, Calif., USA).

The program Primer3 (SourceForge;http://primer3.sourceforqe.net/releases.php) was used to design twointernal primers for the coding region of P24NHZ to identify a plasmidfree of sequencing errors.

Primer KKSC0136fw1: (SEQ ID NO: 23) 5′-CACAGTGCAAGTAGCGAGGA-3′Primer KKSC0136rw1: (SEQ ID NO: 24) 5′-TCGCTACTTGCACTGTGGAG-3′

Combined with the vector primers 8653 and 8654, the spacing of theinternal primers gave overlapping coverage of the entire open readingframe in both directions with an overlap coverage of 500 bp or lessbetween primers. Reads were obtained from the ABI 3730 DNA sequencerused above and the nucleotide bases were called using the KB-basecaller(Applied Biosystems, Foster City, USA) that produces quality scores forthe individual bases in the reads. All reads were assembled to a singlesequence and phredPhrap quality score (http://www.phrap.org).

One plasmid, pKKSC0136-1, in a colony designated E. coli KKSC136-1 wasfree of errors and chosen for expression.

Example 17: Expression of the P24NHZ GH61 Polypeptide Coding Sequencefrom Bulgaria Inquinans 046TR

A. oryzae MT3568 is an amdS (acetamidase) disrupted gene derivative ofAspergillus oryzae JaL355 (WO 2002/40694) in which pyrG auxotrophy wasrestored by inactivating the A. oryzae amdS gene. Protoplasts of A.oryzae MT3568 were prepared according to the method described inEuropean Patent, EP0238023, pages 14-15.

E. coli KKSC136-1 containing pKKSC0136-1 was grown overnight accordingto the manufacturer's instructions (QIAGEN GMBH, Hilden Germany) andplasmid DNA of pKKSC0139-1 was isolated using a Plasmid Midi Kit (QIAGENGMBH, Hilden Germany) according to the manufacturer's instructions. Thepurified plasmid DNA was transformed into Aspergillus oryzae MT3568according to the method described in WO 2005/042735, pages 34-35.Briefly, 8 μl of plasmid DNA representing 3 μg of DNA were added to 100μl of the A. oryzae MT3568 protoplasts. A 250 μl volume of 60% PEGsolution was added and the tubes were gently mixed and incubated at 37°C. for 30 minutes. The mix was added to 10 ml of pre-melted COVE topagarose, which was melted and then temperature equilibrated to 40° C. ina warm water bath before being added to the protoplast mixture. Thecombined mixture was then spread onto two COVE sucrose plates containing10 mM acetamide. The plates were incubated at 37° C. for 4 days. SingleA. oryzae transformed colonies were identified by growth on acetamide asa carbon source. Four A. oryzae transformants were isolated andinoculated into 750 μl of YP+2% glucose medium, 750 μl of YP+2%maltodextrin medium, and DAP4C medium in 96 well deep plates andincubated at 37° C. stationary for 4 days. At the same time the fourtransformants were restreaked on COVE-2 plates.

Culture broth from the Aspergillus oryzae transformants were thenanalyzed for production of the P24GAB GH61 polypeptide by SDS-PAGE usingNUPAGE® 10% Bis-Tris SDS gels (Invitrogen, Carlsbad, Calif., USA)according to the manufacturer. A band at approximately 60 kDa wasobserved for each of the Aspergillus oryzae transformants. One A. oryzaetransformant producing the P24NHZ GH61 polypeptide was designated A.oryzae EXP08201.

Alternatively, a synthetic gene based on the nucleotide sequenceidentified as SEQ ID NO: 7 can be obtained from a number of vendors suchas Gene Art (GENEART AG BioPark, Josef-Engert-Str. 11, 93053,Regensburg, Germany) or DNA 2.0 (DNA2.0, 1430 O'Brien Drive, Suite E,Menlo Park, Calif. 94025, USA). The synthetic gene can be designed toincorporate additional DNA sequences such as restriction sites orhomologous recombination regions to facilitate cloning into anexpression vector.

Using the two synthetic oligonucleotide primers KKSC0136-F andKKSC0136-R described above, a simple PCR reaction can be used to amplifythe full-length open reading frame from the synthetic gene of SEQ ID NO:7. The gene can then be cloned into an expression vector as describedherein and expressed in a host cell as described herein, e.g.,Aspergillus oryzae.

Example 18: Characterization of the P24NHZ GH61 Polypeptide CodingSequence from Bulgaria Inquinans 046TR

The genomic DNA sequence and deduced amino acid sequence of the Bulgariainquinans P24NHZ GH61 polypeptide coding sequence are shown in SEQ IDNO: 7 and SEQ ID NO: 8, respectively. The coding sequence is 969 bpincluding the stop codon with no intervening introns. The encodedpredicted protein is 322 amino acids. Using the SignalP 3.0 program(Bendtsen et al., 2004, supra), a signal peptide of 16 residues waspredicted. The SignalP prediction is in accord with the necessity forhaving a histidine reside at the N-terminus in order for proper metalbinding and hence protein function to occur (See Harris et al., 2010,supra, and Quinlan et al., 2011, supra). The predicted mature proteincontains 306 amino acids with a predicted molecular mass of 31 kDa and apredicted isoelectric point of 3.68. An unusual C terminal extension isobserved at positions 243 to 319 in the GH61 polypeptide. This extensionis not part of the GH61 enzyme module.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, supra) with a gap open penalty of 10, a gap extensionpenalty of 0.5, and the EBLOSUM62 matrix. The alignment showed that thededuced amino acid sequence of the Bulgaria inquinans genomic DNAencoding the P24NHZ GH61 polypeptide shares 63.82% identity to(excluding gaps) to the deduced amino acid sequence of a GH61polypeptide from Acremonium cellulolyticus (GENESEQP:AZF55171)

Example 19: Preparation of Bulgaria Inquinans GH61 Polypeptide

Aspergillus oryzae EXP08201 was cultivated in 1000 ml Erlenmeyer shakeflasks containing 100 ml of YP+2% glucose medium at 26° C. for 4 dayswith agitation at 85 rpm. The broth was filtered using a 0.22 μmEXPRESS™ Plus Membrane (Millipore, Bedford, Mass., USA). A 100 ml volumeof the filtered broth was concentrated to about 10 ml using VIVASPIN® 20(10 kDa MWCO) spin concentrators and centrifuging (Sorvall, LegendRT+Centrifuge, Thermo Scientific, Germany) at 3000 rpm for 15 minuteintervals repeatedly. The total protein content of the GH61 polypeptidewas determined by gel quantitation following quantitative desalting. A 3ml volume of the concentrated GH61 polypeptide broth was desalted andbuffer exchanged into 50 mM sodium acetate pH 5.0 using an ECONO-PAC®10-DG desalting column (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA). Protein concentration was determined by SDS-PAGE using an 8-16%Tris HCl CRITERION STAIN FREE™ gel (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA) and a CRITERION STAIN FREE™ Imaging System(Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Example 20: Effect of the Bulgaria Inquinans GH61 Polypeptide on theHydrolysis of Microcrystalline Cellulose

The Bulgaria inquinans GH61 polypeptide was evaluated for the ability toenhance the hydrolysis of microcrystalline cellulose by Trichodermareesei GH5 endoglucanase II (loaded at 2 mg protein per g cellulose) andAspergillus fumigatus GH3 beta-glucosidase (loaded at 2 mg protein per gcellulose) with and without the addition of 20 mM catechol at 50° C. TheB. inquinans GH61 polypeptide was added at 5 mg protein per g cellulose.The composition of T. reesei GH5 endoglucanase II (loaded at 2 mgprotein per g cellulose) and A. fumigatus GH3 beta-glucosidase (loadedat 2 mg protein per g cellulose) was also run as a control without addedGH61 polypeptide.

The assay was performed as described in Example 11. The 1 ml reactionswith microcrystalline cellulose were conducted for 72 hours in 50 mMammonium acetate pH 5.0 containing 10 μM copper sulfate. All reactionswere performed in triplicate and involved single mixing at the beginningof hydrolysis.

As shown in FIG. 6, hydrolysis of the microcrystalline cellulose by thecomposition of T. reesei GH5 endoglucanase II and A. fumigatus GH3beta-glucosidase without catechol produced similar results as thatobtained with the B. inquinans GH61 polypeptide added to the compositionof T. reesei GH5 endoglucanase II and A. fumigatus GH3 beta-glucosidasewithout catechol. The addition of B. inquinans GH61 polypeptide to thecomposition of T. reesei GH5 endoglucanase II and A. fumigatus GH3beta-glucosidase without catechol did not improve hydrolysis of themicrocrystalline cellulose. However, as shown in FIG. 6, the addition ofB. inquinans GH61 polypeptide to the composition of T. reesei GH5endoglucanase II and A. fumigatus GH3 beta-glucosidase with 20 mMcatechol resulted in a higher degree of glucose production (shown ing/L) compared to the addition of B. inquinans GH61 polypeptide to thecomposition of T. reesei GH5 endoglucanase II and A. fumigatus GH3beta-glucosidase without added catechol and compared to the compositionof T. reesei GH5 endoglucanase II and A. fumigatus GH3 beta-glucosidasewithout GH61 polypeptide and without added catechol. The resultsdemonstrated a 1.17-fold improvement (or 17% increase) in hydrolysis ofthe microcrystalline cellulose by B. inquinans GH61 polypeptide additionto the composition of T. reesei GH5 endoglucanase II and A. fumigatusGH3 beta-glucosidase with catechol compared to without catechol.

Example 21: Pretreated Corn Stover Hydrolysis Assay

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using 1.4 wt. % sulfuric acid at 165°C. and 107 psi for 8 minutes. The water-insoluble solids in thepretreated corn stover (PCS) contained 56.5% cellulose, 4.6%hemicellulose and 28.4% lignin. Cellulose and hemicellulose weredetermined by a two-stage sulfuric acid hydrolysis with subsequentanalysis of sugars by high performance liquid chromatography using NRELStandard Analytical Procedure #002. Lignin was determinedgravimetrically after hydrolyzing the cellulose and hemicellulosefractions with sulfuric acid using NREL Standard Analytical Procedure#003.

Unmilled, unwashed PCS (whole slurry PCS) was prepared by adjusting thepH of the PCS to 5.0 by addition of 10 M NaOH with extensive mixing, andthen autoclaving for 20 minutes at 120° C. The dry weight of the wholeslurry PCS was 29%. Milled unwashed PCS (dry weight 32.35%) was preparedby milling whole slurry PCS in a Cosmos ICMG 40 wet multi-utilitygrinder (EssEmm Corporation, Tamil Nadu, India).

The hydrolysis of PCS was conducted using 2.2 ml deep-well plates(Axygen, Union City, Calif., USA) in a total reaction volume of 1.0 ml.The hydrolysis was performed with 50 mg of insoluble PCS solids per mlof 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfateand various protein loadings of various enzyme compositions (expressedas mg protein per gram of cellulose). Enzyme compositions were preparedand then added simultaneously to all wells in a volume ranging from 50μl to 200 μl, for a final volume of 1 ml in each reaction. The plate wasthen sealed using an ALPS300™ plate heat sealer, mixed thoroughly, andincubated at a specific temperature for 72 hours. All experimentsreported were performed in triplicate.

Following hydrolysis, samples were filtered using a 0.45 μm MULTISCREEN®96-well filter plate and the filtrates were analyzed for sugar contentas described below. When not used immediately, filtered aliquots werefrozen at −20° C. The glucose concentration of the samples diluted in0.005 M H₂SO₄ was measured using a 4.6×250 mm AMINEX® HPX-87H column byelution with 0.05% w/w benzoic acid-0.005 M H₂SO₄ at 65° C. at a flowrate of 0.6 ml per minute, and quantitation by integration of theglucose signal from refractive index detection (CHEMSTATION®, AGILENT®1100 HPLC) calibrated by a pure glucose standard. The resultant glucoseequivalents were used to calculate the percentage of celluloseconversion for each reaction.

The measured glucose concentrations were adjusted for the appropriatedilution factor. The net concentrations of enzymatically-producedglucose from the milled unwashed PCS were determined by adjusting themeasured glucose concentrations for corresponding background glucoseconcentrations in unwashed PCS at a zero time point. All HPLC dataprocessing was performed using MICROSOFT EXCEL™ software.

The degree of cellulose conversion to glucose was calculated using thefollowing equation: % conversion=(glucose concentration/glucoseconcentration in a limit digest)×100. In order to calculate %conversion, a 100% conversion point was set based on a cellulase control(100 mg of Trichoderma reesei cellulase per gram cellulose), and allvalues were divided by this number and then multiplied by 100.Triplicate data points were averaged and standard deviation wascalculated.

Example 22: Preparation of an Enzyme Composition

The Aspergillus fumigatus GH7A cellobiohydrolase I (SEQ ID NO: 25 [DNAsequence] and SEQ ID NO: 26 [deduced amino acid sequence]) was preparedrecombinantly in Aspergillus oryzae as described in WO 2011/057140. Thefiltered broth of the A. fumigatus cellobiohydrolase I was concentratedand buffer exchanged using a tangential flow concentrator (Pall Filtron,Northborough, Mass., USA) equipped with a 10 kDa polyethersulfonemembrane (Pall Filtron, Northborough, Mass., USA) with 20 mM Tris-HCl pH8.0. The desalted broth of the A. fumigatus cellobiohydrolase I wasloaded onto a Q SEPHAROSE® ion exchange column (GE Healthcare,Piscataway, N.J., USA) equilibrated in 20 mM Tris-HCl pH 8 and elutedusing a linear 0 to 1 M NaCl gradient. Fractions were collected andfractions containing the cellobiohydrolase I were pooled based onSDS-PAGE analysis using 8-16% CRITERION® Stain-free SDS-PAGE gels(Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

The Aspergillus fumigatus GH6A cellobiohydrolase II (SEQ ID NO: 27 [DNAsequence] and SEQ ID NO: 28 [deduced amino acid sequence]) was preparedrecombinantly in Aspergillus oryzae as described in WO 2011/057140. Thefiltered broth of the A. fumigatus cellobiohydrolase II was bufferexchanged into 20 mM Tris pH 8.0 using a 400 ml SEPHADEX™ G-25 column(GE Healthcare, United Kingdom). The fractions were pooled and adjustedto 1.2 M ammonium sulphate-20 mM Tris pH 8.0. The equilibrated proteinwas loaded onto a PHENYL SEPHAROSE™ 6 Fast Flow column (high sub) (GEHealthcare, Piscataway, N.J., USA) equilibrated in 20 mM Tris pH 8.0with 1.2 M ammonium sulphate, and bound proteins were eluted with 20 mMTris pH 8.0 with no ammonium sulphate. The fractions were pooled.

The Trichoderma reesei GH5 endoglucanase II was prepared as described inExample 9.

The Aspergillus fumigatus GH10 xylanase (xyn3) (SEQ ID NO: 29 [DNAsequence] and SEQ ID NO: 30 [deduced amino acid sequence]) was preparedrecombinantly according to WO 2006/078256 using Aspergillus oryzae BECh2(WO 2000/39322) as a host. The filtered broth of the A. fumigatusxylanase was desalted and buffer-exchanged into 50 mM sodium acetate pH5.0 using a HIPREP® 26/10 Desalting Column (GE Healthcare, Piscataway,N.J., USA).

The Aspergillus fumigatus NN055679 Cel3A beta-glucosidase was preparedas described in Example 10.

The Aspergillus fumigatus NN051616 GH3 beta-xylosidase (SEQ ID NO: 31[DNA sequence] and SEQ ID NO: 32 [deduced amino acid sequence]) wasprepared recombinantly in Aspergillus oryzae as described in WO2011/057140. The filtered broth of the A. fumigatus beta-xylosidase wasdesalted and buffer-exchanged into 50 mM sodium acetate pH 5.0 using aHIPREP® 26/10 Desalting Column.

The protein concentration for each of the monocomponents described abovewas determined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard. An enzyme composition wasprepared composed of each monocomponent as follows: 37% Aspergillusfumigatus Cel7A cellobiohydrolase I, 25% Aspergillus fumigatus Cel6Acellobiohydrolase II, 10% Trichoderma reesei GH5 endoglucanase II, 5%Aspergillus fumigatus GH10 xylanase, 5% Aspergillus fumigatusbeta-glucosidase, and 3% Aspergillus fumigatus beta-xylosidase. Theenzyme composition is designated herein as “cellulolytic enzymecomposition”.

Example 23: Effect of the Bulgaria Inquinans GH61 Polypeptide on theHydrolysis of Milled Unwashed PCS by a Cellulolytic Enzyme Composition

The Bulgaria inquinans GH61 polypeptide was evaluated for the ability toenhance the hydrolysis of milled unwashed PCS (Example 21) by thecellulolytic enzyme composition (Example 22) at 2.55 mg total proteinper g cellulose at 50° C., 55° C., 60° C., and 65° C. The Bulgariainquinans GH61 polypeptide was added at 0.45 mg protein per g cellulose.The cellulolytic enzyme composition was also run without added GH61polypeptide at 3.0 mg protein per g cellulose.

The assay was performed as described in Example 21. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

As shown in FIG. 7, the cellulolytic enzyme composition that includedthe B. inquinans GH61 polypeptide significantly outperformed thecellulolytic enzyme composition (2.55 mg protein per g cellulose and 3.0mg protein per g cellulose) without GH61 polypeptide. The degree ofcellulose conversion to glucose for the B. inquinans GH61 polypeptideadded to the cellulolytic enzyme composition was significantly higherthan the cellulolytic enzyme composition without added GH61 polypeptideat 50° C., 55° C., 60° C., and 65° C., especially at 50° C., 55° C., and60° C.

The present invention is further described by the following numberedparagraphs:

[1] An isolated polypeptide having cellulolytic enhancing activity,selected from the group consisting of: (a) a polypeptide having at least60% sequence identity to the mature polypeptide of SEQ ID NO: 6, atleast 65% sequence identity to the mature polypeptide of SEQ ID NO: 8,at least 75% sequence identity to the mature polypeptide of SEQ ID NO:2, or at least 80% sequence identity to the mature polypeptide of SEQ IDNO: 4; (b) a polypeptide encoded by a polynucleotide that hybridizesunder at least high stringency conditions with the mature polypeptidecoding sequence of SEQ ID NO: 1 or the cDNA sequence thereof, the maturepolypeptide of SEQ ID NO: 3 or the cDNA sequence thereof, the maturepolypeptide of SEQ ID NO: 5 or the cDNA sequence thereof, or the maturepolypeptide of SEQ ID NO: 7; or the full-length complement thereof; (c)a polypeptide encoded by a polynucleotide having at least 60% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 5 orthe cDNA sequence thereof, at least 65% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 7, at least 75% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1 orthe cDNA sequence thereof, or at least 80% sequence identity to themature polypeptide coding sequence of SEQ ID NO: 3 or the cDNA sequencethereof; (d) a variant of the mature polypeptide of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, or SEQ ID NO: 8 comprising a substitution,deletion, and/or insertion at one or more positions; and (e) a fragmentof the polypeptide of (a), (b), (c), or (d) that has cellulolyticenhancing activity.

[2] The polypeptide of paragraph 1, having at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide of SEQ ID NO: 6; at least 65%, at least 70%, at least 75%,at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to the mature polypeptide of SEQ ID NO: 8; atleast 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to the mature polypeptide of SEQ IDNO: 2; or at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to the mature polypeptide of SEQ IDNO: 4.

[3] The polypeptide of paragraph 1, which is encoded by a polynucleotidethat hybridizes under high or very high stringency conditions with themature polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequencethereof, the mature polypeptide coding sequence of SEQ ID NO: 3 or thecDNA sequence thereof, the mature polypeptide coding sequence of SEQ IDNO: 5 or the cDNA sequence thereof, or the mature polypeptide codingsequence of SEQ ID NO: 7; or the full-length complement thereof.

[4] The polypeptide of paragraph 1, which is encoded by a polynucleotidehaving at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 5 or the cDNA sequence thereof; at least 65%, at least 70%, at least75%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to the mature polypeptide coding sequenceof SEQ ID NO: 7; at least 75%, at least 80%, at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequencethereof; or at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 3 or the cDNA sequence thereof.

[5] The polypeptide of any of paragraphs 1-4, comprising or consistingof SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8 or themature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQID NO: 8.

[6] The polypeptide of paragraph 5, wherein the mature polypeptide isamino acids 20 to 316 of SEQ ID NO: 2, amino acids 20 to 231 of SEQ IDNO: 4, amino acids 20 to 254 of SEQ ID NO: 6, or amino acids 17 to 322of SEQ ID NO: 8.

[7] The polypeptide of paragraph 1, which is a variant of the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8comprising a substitution, deletion, and/or insertion at one or morepositions.

[8] The polypeptide of any of paragraphs 1-7, which is a fragment of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, wherein thefragment has cellulolytic enhancing activity.

[9] An isolated polypeptide comprising a catalytic domain selected fromthe group consisting of: (a) a catalytic domain having at least 80%sequence identity to amino acids 20 to 245 of SEQ ID NO: 2; (b) acatalytic domain encoded by a polynucleotide that hybridizes under atleast very high stringency conditions with nucleotides 58 to 1122 of SEQID NO: 1; the cDNA sequence thereof; or the full-length complementthereof; (c) a catalytic domain encoded by a polynucleotide having atleast 80% sequence identity to nucleotides 58 to 1122 of SEQ ID NO: 1 orthe cDNA sequence thereof; (d) a variant of amino acids 20 to 245 of SEQID NO: 2 comprising a substitution, deletion, and/or insertion at one ormore positions; and (e) a fragment of the catalytic domain of (a), (b),(c), or (d) that has cellulolytic enhancing activity.

[10] The polypeptide of paragraph 9, further comprising a cellulosebinding domain.

[11] An isolated polypeptide comprising a cellulose binding domainoperably linked to a catalytic domain, wherein the binding domain isselected from the group consisting of: (a) a cellulose binding domainhaving at least 80% sequence identity to amino acids 284 to 316 of SEQID NO: 2; (b) a cellulose binding domain encoded by a polynucleotidethat hybridizes under at least very high stringency conditions withnucleotides 1237 to 1458 of SEQ ID NO: 1; the cDNA sequence thereof; orthe full-length complement thereof; (c) a cellulose binding domainencoded by a polynucleotide having at least 80% sequence identity tonucleotides 1237 to 1458 of SEQ ID NO: 1 or the cDNA sequence thereof;(d) a variant of amino acids 284 to 316 of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more positions; and(e) a fragment of the cellulose binding domain of (a), (b), (c), or (d)that has binding activity.

[12] The polypeptide of paragraph 11, wherein the catalytic domain isobtained from a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase,lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase, xylanase, or beta-xylosidase.

[13] A composition comprising the polypeptide of any of paragraphs 1-12.

[14] An isolated polynucleotide encoding the polypeptide of any ofparagraphs 1-12.

[15] A nucleic acid construct or expression vector comprising thepolynucleotide of paragraph 14 operably linked to one or more controlsequences that direct the production of the polypeptide in an expressionhost.

[16] A recombinant host cell comprising the polynucleotide of paragraph14 operably linked to one or more control sequences that direct theproduction of the polypeptide.

[17] A method of producing the polypeptide of any of paragraphs 1-12,comprising: cultivating a cell, which in its wild-type form produces thepolypeptide, under conditions conducive for production of thepolypeptide.

[18] The method of paragraph 17, further comprising recovering thepolypeptide.

[19] A method of producing a polypeptide having cellulolytic enhancingactivity, comprising: cultivating the host cell of paragraph 16 underconditions conducive for production of the polypeptide.

[20] The method of paragraph 19, further comprising recovering thepolypeptide.

[21] A transgenic plant, plant part or plant cell transformed with apolynucleotide encoding the polypeptide of any of paragraphs 1-12.

[22] A method of producing a polypeptide having cellulolytic enhancingactivity, comprising: cultivating the transgenic plant or plant cell ofparagraph 21 under conditions conducive for production of thepolypeptide.

[23] The method of paragraph 22, further comprising recovering thepolypeptide.

[24] A method of producing a mutant of a parent cell, comprisinginactivating a polynucleotide encoding the polypeptide of any ofparagraphs 1-12, which results in the mutant producing less of thepolypeptide than the parent cell.

[25] A mutant cell produced by the method of paragraph 24.

[26] The mutant cell of paragraph 25, further comprising a gene encodinga native or heterologous protein.

[27] A method of producing a protein, comprising: cultivating the mutantcell of paragraph 25 or 26 under conditions conducive for production ofthe protein.

[28] The method of paragraph 27, further comprising recovering theprotein.

[29] A double-stranded inhibitory RNA (dsRNA) molecule comprising asubsequence of the polynucleotide of paragraph 14, wherein optionallythe dsRNA is an siRNA or an miRNA molecule.

[30] The double-stranded inhibitory RNA (dsRNA) molecule of paragraph29, which is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or moreduplex nucleotides in length.

[31] A method of inhibiting the expression of a polypeptide havingcellulolytic enhancing activity in a cell, comprising administering tothe cell or expressing in the cell the double-stranded inhibitory RNA(dsRNA) molecule of paragraph 29 or 30.

[32] A cell produced by the method of paragraph 31.

[33] The cell of paragraph 32, further comprising a gene encoding anative or heterologous protein.

[34] A method of producing a protein, comprising: cultivating the cellof paragraph 32 or 33 under conditions conducive for production of theprotein.

[35] The method of paragraph 35, further comprising recovering theprotein.

[36] An isolated polynucleotide encoding a signal peptide comprising orconsisting of amino acids 1 to 19 of SEQ ID NO: 2, amino acids 1 to 19of SEQ ID NO: 4, amino acids 1 to 19 of SEQ ID NO: 6, or amino acids 1to 16 of SEQ ID NO: 8.

[37] A nucleic acid construct or expression vector comprising a geneencoding a protein operably linked to the polynucleotide of paragraph36, wherein the gene is foreign to the polynucleotide encoding thesignal peptide.

[38] A recombinant host cell comprising a gene encoding a proteinoperably linked to the polynucleotide of paragraph 36, wherein the geneis foreign to the polynucleotide encoding the signal peptide.

[39] A method of producing a protein, comprising: cultivating arecombinant host cell comprising a gene encoding a protein operablylinked to the polynucleotide of paragraph 36, wherein the gene isforeign to the polynucleotide encoding the signal peptide, underconditions conducive for production of the protein.

[40] The method of paragraph 39, further comprising recovering theprotein.

[41] A process for degrading a cellulosic material, comprising: treatingthe cellulosic material with an enzyme composition in the presence ofthe polypeptide having cellulolytic enhancing activity of any ofparagraphs 1-12.

[42] The process of paragraph 41, wherein the cellulosic material ispretreated.

[43] The process of paragraph 41 or 42, wherein the enzyme compositioncomprises one or more enzymes selected from the group consisting of acellulase, a polypeptide having cellulolytic enhancing activity, ahemicellulase, a CIP, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.

[44] The process of paragraph 43, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[45] The process of paragraph 43, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[46] The process of any of paragraphs 41-45, further comprisingrecovering the degraded cellulosic material.

[47] The process of paragraph 46, wherein the degraded cellulosicmaterial is a sugar.

[48] The process of paragraph 47, wherein the sugar is selected from thegroup consisting of glucose, xylose, mannose, galactose, and arabinose.

[49] A process for producing a fermentation product, comprising: (a)saccharifying a cellulosic material with an enzyme composition in thepresence of the polypeptide having cellulolytic enhancing activity ofany of paragraphs 1-12; (b) fermenting the saccharified cellulosicmaterial with one or more fermenting microorganisms to produce thefermentation product; and (c) recovering the fermentation product fromthe fermentation.

[50] The process of paragraph 49, wherein the cellulosic material ispretreated.

[51] The process of paragraph 49 or 50, wherein the enzyme compositioncomprises the enzyme composition comprises one or more enzymes selectedfrom the group consisting of a cellulase, a polypeptide havingcellulolytic enhancing activity, a hemicellulase, a CIP, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.

[52] The process of paragraph 51, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[53] The process of paragraph 51, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[54] The process of any of paragraphs 49-53, wherein steps (a) and (b)are performed simultaneously in a simultaneous saccharification andfermentation.

[55] The process of any of paragraphs 49-54, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.

[56] A process of fermenting a cellulosic material, comprising:fermenting the cellulosic material with one or more fermentingmicroorganisms, wherein the cellulosic material is saccharified with anenzyme composition in the presence of the polypeptide havingcellulolytic enhancing activity of any of paragraphs 1-12.

[57] The process of paragraph 56, wherein the fermenting of thecellulosic material produces a fermentation product.

[58] The process of paragraph 57, further comprising recovering thefermentation product from the fermentation.

[59] The process of paragraph 57 or 58, wherein the fermentation productis an alcohol, an alkane, a cycloalkane, an alkene, an amino acid, agas, isoprene, a ketone, an organic acid, or polyketide.

[60] The process of any of paragraphs 56-59, wherein the cellulosicmaterial is pretreated before saccharification.

[61] The process of any of paragraphs 56-60, wherein the enzymecomposition comprises one or more enzymes selected from the groupconsisting of a cellulase, a polypeptide having cellulolytic enhancingactivity, a hemicellulase, a CIP, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.

[62] The process of paragraph 61, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[63] The process of paragraph 61, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[64] A whole broth formulation or cell culture composition comprisingthe polypeptide of any of paragraphs 1-12.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

What is claimed is:
 1. A nucleic acid construct, comprising apolynucleotide encoding a GH61 polypeptide having cellulolytic enhancingactivity, wherein the polynucleotide is operably linked to one or moreheterologous control sequences that direct the production of the GH61polypeptide in a recombinant host cell, and wherein the GH61 polypeptidehaving cellulolytic enhancing activity is selected from the groupconsisting of: (a) a GH61 polypeptide having at least 95% sequenceidentity to the mature polypeptide of SEQ ID NO: 8; (b) a GH61polypeptide encoded by a polynucleotide that hybridizes under very highstringency conditions with the full-length complement of the maturepolypeptide coding sequence of SEQ ID NO: 7 or the cDNA sequence of themature polypeptide coding sequence of SEQ ID NO: 7, wherein the veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, for 12 to 24 hours,and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 70°C.; and (c) a GH61 polypeptide encoded by a polynucleotide having atleast 99% sequence identity to the mature polypeptide coding sequence ofSEQ ID NO: 7 or the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO:
 7. 2. The nucleic acid construct of claim 1,wherein the GH61 polypeptide has at least 95% sequence identity to themature polypeptide of SEQ ID NO:
 8. 3. The nucleic acid construct ofclaim 1, wherein the GH61 polypeptide has at least 96% sequence identityto the mature polypeptide of SEQ ID NO:
 8. 4. The nucleic acid constructof claim 1, wherein the GH61 polypeptide has at least 97% sequenceidentity to the mature polypeptide of SEQ ID NO:
 8. 5. The nucleic acidconstruct of claim 1, wherein the GH61 polypeptide has at least 98%sequence identity to the mature polypeptide of SEQ ID NO:
 8. 6. Thenucleic acid construct of claim 1, wherein the GH61 polypeptide has atleast 99% sequence identity to the mature polypeptide of SEQ ID NO: 8.7. The nucleic acid construct of claim 1, wherein the GH61 polypeptidecomprises SEQ ID NO: 8 or the mature polypeptide of SEQ ID NO:
 8. 8. Thenucleic acid construct of claim 1, wherein the GH61 polypeptidecomprises amino acids 17 to 322 of SEQ ID NO:
 8. 9. A recombinant hostcell transformed with the nucleic acid construct of claim
 1. 10. Amethod of producing a GH61 polypeptide having cellulolytic enhancingactivity, comprising: (a) cultivating the recombinant host cell of claim9 under conditions conducive for production of the polypeptide, andoptionally (b) recovering the polypeptide.
 11. A recombinant host cell,transformed with a nucleic acid construct comprising a polynucleotideencoding a GH61 polypeptide having cellulolytic enhancing activity,wherein the polynucleotide is operably linked to one or more controlsequences that direct the production of the GH61 polypeptide in therecombinant host cell, wherein the GH61 polypeptide having cellulolyticenhancing activity is heterologous to the recombinant host cell, andwherein the GH61 polypeptide having cellulolytic enhancing activity isselected from the group consisting of: (a) a GH61 polypeptide having atleast 95% sequence identity to the mature polypeptide of SEQ ID NO: 8;(b) a GH61 polypeptide encoded by a polynucleotide that hybridizes undervery high stringency conditions with the full-length complement of themature polypeptide coding sequence of SEQ ID NO: 7 or the cDNA sequenceof the mature polypeptide coding sequence of SEQ ID NO: 7, wherein thevery high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, for 12 to 24 hours,and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 70°C.; and (c) a GH61 polypeptide encoded by a polynucleotide having atleast 99% sequence identity to the mature polypeptide coding sequence ofSEQ ID NO: 7 or the cDNA sequence of the mature polypeptide codingsequence of SEQ ID NO:
 7. 12. The recombinant host cell of claim 11,wherein the GH61 polypeptide has at least 95% sequence identity to themature polypeptide of SEQ ID NO:
 8. 13. The recombinant host cell ofclaim 11, wherein the GH61 polypeptide has at least 96% sequenceidentity to the mature polypeptide of SEQ ID NO:
 8. 14. The recombinanthost cell of claim 11, wherein the GH61 polypeptide has at least 97%sequence identity to the mature polypeptide of SEQ ID NO:
 8. 15. Therecombinant host cell of claim 11, wherein the GH61 polypeptide has atleast 98% sequence identity to the mature polypeptide of SEQ ID NO: 8.16. The recombinant host cell of claim 11, wherein the GH61 polypeptidehas at least 99% sequence identity to the mature polypeptide of SEQ IDNO:
 8. 17. The recombinant host cell of claim 11, wherein the GH61polypeptide comprises SEQ ID NO: 8 or the mature polypeptide of SEQ IDNO:
 8. 18. The recombinant host cell of claim 11, wherein the GH61polypeptide comprises amino acids 17 to 322 of SEQ ID NO:
 8. 19. Amethod of producing a GH61 polypeptide having cellulolytic enhancingactivity, comprising: (a) cultivating the recombinant host cell of claim11 under conditions conducive for production of the polypeptide, andoptionally (b) recovering the polypeptide.
 20. A transgenic plant, plantpart or plant cell transformed with the nucleic acid construct ofclaim
 1. 21. A method of producing a GH61 polypeptide havingcellulolytic enhancing activity, comprising: (a) cultivating thetransgenic plant or plant cell of claim 20 under conditions conducivefor production of the polypeptide, and optionally (b) recovering thepolypeptide.